WO2003012126A2 - Epoxide hydrolases, nucleic acids encoding them and methods for making and using them - Google Patents
Epoxide hydrolases, nucleic acids encoding them and methods for making and using them Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- This invention relates to molecular and cellular biology and biochemistry.
- the invention is directed to polypeptides having an epoxide hydrolase activity, polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides.
- the polypeptides of the invention can be used as epoxide hydrolases to catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols.
- Epoxide hydrolases catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols.
- Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolyzed epoxide in nonracemic form, enantioconvergent processes are possible. These are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane, see, e.g., Steinreiber (2001) Curr.
- Microsomal epoxide hydrolases are biotransformation enzymes that catalyze the conversion of abroad array of xenobiotic epoxide substrates to more polar diol metabolites, see, e.g., Omiecinski (2000) Toxicol. Lett. 112-113:365-370.
- Microsomal epoxide hydrolases catalyze the addition of water to epoxides in a two-step reaction involving initial attack of an active site carboxylate on the oxirane to give an ester intermediate followed by hydrolysis of the ester.
- Soluble epoxide hydrolase play a role in the biosynthesis of inflammation mediators, see, e.g., Morisseau (1999) Proc. Natl. Acad. Sci. USA 96:8849-8854.
- Chiral molecules including alcohols, ⁇ -hydroxy acids and epoxides, are important for the synthesis of pharmaceuticals, agrochemicals, as well as many fine chemicals.
- a major challenge in modern organic chemistry is to generate such compounds in high yields, with high stereo- and regioselectivities.
- Enantiopure epoxides are versatile synthons for the synthesis of numerous pharmaceuticals, agrochemicals and other high value compounds.
- EHs epoxide hydrolases
- the selective hydrolysis of a racemic epoxide can generate both the corresponding diols and the unreacted epoxides with high enantiomeric excess (ee) values.
- the following significant limitations urgently need to be overcome: (1) the number of enzymes available is small; and (2) the scope of substrates is limited.
- Enzymes for example, have a number of remarkable advantages, which can overcome these problems in catalysis: they act on single functional groups, they distinguish between similar functional groups on a single molecule, and they distinguish between enantiomers. Moreover, they are biodegradable and function at very low mole fractions in reaction mixtures. Because of their chemo-, regio- and stereospecificity, enzymes present a unique opportunity to optimally achieve desired selective transformations. These are often extremely difficult to duplicate chemically, especially in single-step reactions.
- Enzymes have evolved by selective pressure to perform very specific biological functions within the milieu of a living organism, under conditions of temperature, pH and salt concentration. For the most part, the non-DNA modifying enzyme activities thus far identified have been isolated from mesophilic organisms, which represent a very small fraction of the available phylogenetic diversity. The dynamic field of biocatalysis takes on a new dimension with the help of enzymes isolated from microorganisms that thrive in extreme environments.
- such enzymes must function at temperatures above 100°C in terrestrial hot springs and deep sea thermal vents, at temperatures below 0°C in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge.
- the invention provides not only a source of materials for the development of biologies, therapeutics, and enzymes for industrial applications, but also provides a new materials for further processing by, for example, directed evolution and mutagenesis to develop molecules or polypeptides modified for particular activity, specificity or conditions.
- the invention can be used to obtain and identify polynucleotides and related sequence specific information from, for example, infectious microorganisms present in the environment such as, for example, in the gut of various macroorganisms.
- Identifying novel enzymes in an environmental sample is one solution to this problem.
- the invention provides methods, compositions and sources for the development of biologies, diagnostics, therapeutics, and compositions for industrial applications.
- Chiral epoxides and diols are key building blocks for the synthesis of pharmaceuticals.
- the epoxide group is readily transformed into a wide range of derivatives by acid or base-catalyzed ring opening reactions, while the diols similarly can be converted into a diverse range of structures.
- Epoxides have broad applications in areas such as anticancer agents, beta-blockers, beta agonists, antivirals, antifungals, and antibacterials.
- Opportunities for chiral epoxides exist in both the small synthon area, including C-3 and C-4 units, and the advanced chemical intermediate area for pharmaceuticals.
- the C-3 synthons are of maj or significance because they are used in the processes of many pharmaceuticals and can also lead to a wide range of downstream products.
- Glycidols (S-(l), and R-(2)) are the leading chiral epoxides among representative C-3 synthons shown in Figure 2.
- R-glycidol is used as a building block for atenolol (an antihypertensive drug) and S-glycidol leads to R-glycidyl butyrate (7), an important synthon in the synthesis of oxazolidinone antibiotics.
- Oxazolidinones represent a relatively new class of antibiotics and currently there are over 40 at various stages of clinical development.
- Epoxide 8 leads to the production of over 30 other chiral epoxides that are not readily available.
- Epoxide 10 is used in the production of saquinavir, an antiviral drug, while its diastereoisomer 11 is used in the synthesis of amprenavir, another antiviral drug ( Figure 3).
- the mixture of the two compounds can be prepared from phenylalanine through an alkene intermediate.
- Another epoxide, 12, is the building block for the synthesis of two anticancer drugs, docetaxel and paclitaxel ( Figure 4).
- the Sharpless approach suffers a significant drawback as the alkenes must have hydroxyl functionality in the allylic position.
- the asymmetric epoxidation methodology developed by Jacobsen and Katsuki, ** (Jacobsen, E. N. Asymmetric catalytic epoxidation of unfunctionalized olefins. Ju Catalytic Asymmetric Synthesis; Ojima, I. Ed.; VCH: New York, 1993; pp. 159-202; and Katsuki, T. Coord. Chem. Rev. 1995, 140, 189-214) which uses optically active (salen)manganese(III) complexes, does not require allylic alcohols.
- Epoxides may be produced indirectly from alkenes by haloperoxidases, via initial halohydrin formation and subsequent ring closure.
- haloperoxidases via initial halohydrin formation and subsequent ring closure.
- niger EH prefers styrene-oxide types of substrates, and hydrolyzes i?-enantiomers in all the transformations in Figure 5.
- high concentrations of enzymes either whole cells or crude extract
- substrate concentrations had to be used because of the enzymes' low catalytic efficiency.
- Novel EHs need to be discovered to offer complementary enantioselectivity (for example, those that recognize S-enantiomers).
- EHs suitable for large-scale preparation of different types of epoxides also need to be discovered. Equally important is to improve the stereoselectivity and activity of the existing and new EHs using protein engineering technologies.
- the invention provides isolated or recombinant nucleic acids comprising a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:ll, SEQ JJ NO:13, SEQ ID NO:15, SEQ ID NO-.17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ JJD NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:53, SEQ JD NO:57, SEQ JD NO:59, SEQ JD NO:61, SEQ JD NO:63, SEQ TD NO:67, SEQ JD NO:69, SEQ ID NO:71, SEQ JD NO:73, SEQ TD NO:75, SEQ TD NO:77, or SEQ ID NO:79 over a region of at least about 100 residue
- the isolated or recombinant nucleic acids comprise a nucleic acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more sequence identity to SEQ ID NO:l, SEQ ID NO:3, SEQ TD NO:5, SEQ ID NO:7, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ JD NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ JD NO:41, SEQ JD NO:43, SEQ ID NO:45, SEQ JD NO:47, SEQ JD NO:53, SEQ JD NO:57, SEQ JD NO:59, SEQ TD NO:61, SEQ JD NO:63, SEQ JD NO:67, SEQ TD NO:69, SEQ ID NO:71, SEQ ID NO:73
- the isolated or recombinant nucleic acid comprises a nucleic acid sequence having at least 99% sequence identity to SEQ TD NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ TD NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, or SEQ TD NO:79 over a region of at least about 100 residues.
- the isolated or recombinant nucleic acid comprises a nucleic acid having a sequence as set forth in SEQ JD NO:l, a nucleic acid having a sequence as set forth in SEQ JD NO:3, a nucleic acid having a sequence as set forth in SEQ ID NO:5, a nucleic acid having a sequence as set forth in SEQ ID NO:7, a nucleic acid having a sequence as set forth in SEQ ID NO:9, a nucleic acid having a sequence as set forth in SEQ ID NO:l 1, a nucleic acid having a sequence as set forth in SEQ ID NO: 13, a nucleic acid having a sequence as set forth in SEQ ID NO: 15, a nucleic acid having a sequence as set forth in SEQ JD NO: 17, a nucleic acid having a sequence as set forth in SEQ TD NO:19, a nucleic acid having a sequence as set forth in SEQ TD NO:21, a nucleic acid having a sequence
- the nucleic acid sequence encodes a polypeptide comprising a polypeptide having a sequence as set forth in SEQ ID NO:2, a polypeptide having a sequence as set forth in SEQ ID NO:4, a polypeptide having a sequence as set forth in SEQ ID NO:6, a polypeptide having a sequence as set forth in SEQ TD NO:8, a polypeptide having a sequence as set forth in SEQ ID NO: 10, a polypeptide having a sequence as set forth in SEQ ID NO: 12, a polypeptide having a sequence as set forth in SEQ ID NO:14, a polypeptide having a sequence as set forth in SEQ ID NO:16, a polypeptide having a sequence as set forth in SEQ ID NO: 18, a polypeptide having a sequence as set forth in SEQ TD NO:20, a polypeptide having a sequence as set forth in SEQ ID NO:22, a polypeptide having a sequence as set forth in SEQ ID NO:24,
- the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d "nr pataa" -F F, and all other options are set to default.
- the epoxide hydrolase activity comprises catalyzing the addition of water to an oxirane compound.
- the epoxide hydrolase activity can further comprise formation of a corresponding diol.
- the epoxide hydrolase activity can further comprise formation of an enantiomerically enriched epoxide.
- the oxirane compound can comprise an epoxide or arene oxide.
- the oxirane compound or the corresponding diol can be optically active.
- the oxirane compound or the corresponding diol is enantiomerically pure.
- the epoxide hydrolase activity can be enantioselective.
- the epoxide hydrolase activity is thermostable.
- the polypeptide can retain an epoxide hydrolase activity under conditions comprising a temperature range of between about 37°C to about 70°C.
- the epoxide hydrolase activity is thermotolerant.
- the polypeptide can retain an epoxide hydrolase activity after exposure to a temperature in the range from greater than 37°C to about 90°C.
- the polypeptide retains an epoxide hydrolase activity after exposure to a temperature in the range from greater than 37°C to about 50°C.
- the invention provides an isolated or recombinant nucleic acid, wherein the nucleic acid comprises a sequence that hybridizes under stringent conditions to a nucleic acid comprising a sequence as set forth in SEQ ID NO:l, a sequence as set forth in SEQ ID NO:3, a sequence as set forth in SEQ ID NO:5, a sequence as set forth in SEQ ID NO:7, a sequence as set forth in SEQ ID NO:9, a sequence as set forth in SEQ TD NO:l 1, a sequence as set forth in SEQ ID NO: 13, a sequence as set forth in SEQ ID NO:
- sequence as set forth in SEQ ID NO: 15 a sequence as set forth in SEQ ID NO: 17, a sequence as set forth in SEQ ID NO: 19 a sequence as set forth in SEQ ID NO:21, a sequence as set forth in SEQ ID NO:23 a sequence as set forth in SEQ ID NO:25, a sequence as set forth in SEQ ID NO:27 a sequence as set forth in SEQ ID NO:29, a sequence as set forth in SEQ ID NO:31 a sequence as set forth in SEQ ID NO:33, a sequence as set forth in SEQ ID NO:35 a sequence as set forth in SEQ ID NO:37, a sequence as set forth in SEQ JD NO:39 a sequence as set forth in SEQ JD NO:41, a sequence as set forth in SEQ JD NO:43 a sequence as set forth in SEQ ID NO:45, a sequence as set forth in SEQ ID NO:47 a sequence as set forth in SEQ ID NO:49, a sequence as set forth in SEQ ID NO
- the nucleic acid is at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400, or more residues, or, the full length of the gene or transcript.
- the stringent conditions include a wash step comprising a wash in 0.2X SSC at a temperature of about 65°C for about 15 minutes.
- the invention provides a nucleic acid probe for identifying a nucleic acid encoding a polypeptide with an epoxide hydrolase activity, wherein the probe comprises at least 10 consecutive bases of a sequence as set forth in SEQ ID NO:l, a sequence as set forth in SEQ ID NO:3, a sequence as set forth in SEQ ID NO:5, a sequence as set forth in SEQ ID NO:7, a sequence as set forth in SEQ ID NO:9, a sequence as set forth in SEQ ID NO:l 1, a sequence as set forth in SEQ ID NO: 13, a sequence as set forth in SEQ ID NO: 15, a sequence as set forth in SEQ ID NO: 17, a sequence as set forth in SEQ ID NO:19, a sequence as set forth in SEQ ID NO:21, a sequence as set forth in SEQ TD NO:23, a sequence as set forth in SEQ ID NO:25, a sequence as set forth in SEQ ID NO:27, a sequence as set forth in SEQ ID NO:29
- the invention provides nucleic acid probes for identifying a nucleic acid encoding a polypeptide having an epoxide hydrolase activity, wherein the probe can comprise a nucleic acid of the invention, e.g., a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:l 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ TD NO:31, SEQ ED NO:33, SEQ ID NO:35, SEQ ID NO:41, SEQ TD NO:43, SEQ TD NO:45, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:59, SEQ TD NO:61, SEQ ID NO:63, SEQ TD NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ
- the probe comprises an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a nucleic acid of the invention, e.g., a nucleic acid sequence as set forth in SEQ ID NO:l, or a subsequence thereof, a sequence as set forth in SEQ ID NO:3, or a subsequence thereof, a sequence as set forth in SEQ ID NO:5, or a subsequence thereof, a sequence as set forth in SEQ ID NO:7, or a subsequence thereof, a sequence as set forth in SEQ ID NO:9, or a subsequence thereof, a sequence as set forth in SEQ ID NO:l 1, or a subsequence thereof, a sequence as set forth in SEQ ID NO: 13, or a subsequence thereof, a sequence as set forth in SEQ ID NO: 15, or a subsequence thereof
- the probe can comprise a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a region of at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400, or more residues of a nucleic acid comprising a sequence as set forth in SEQ ID NO:l, or a subsequence thereof, a sequence as set forth in SEQ ID NO:3, or a subsequence thereof, a sequence as set forth in SEQ ID NO:5, or a subsequence thereof, a sequence as set forth in SEQ ID NO:7, or a subsequence thereof, a sequence as set forth in SEQ ID NO:9, or a subsequence thereof, a sequence as set forth
- the invention provides an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having a epoxide hydrolase activity, wherein the primer pair is capable of amplifying a nucleic acid of the invention, e.g., a sequence as set forth in SEQ ED NO:l, or a subsequence thereof, a sequence as set forth in SEQ ID NO:3, or a subsequence thereof, a sequence as set forth in SEQ ID NO:5, or a subsequence thereof, a sequence as set forth in SEQ ID NO:7, or a subsequence thereof, a sequence as set forth in SEQ ID NO:9, or a subsequence thereof, a sequence as set forth in SEQ ID NO:l 1, or a subsequence thereof, a sequence as set forth in SEQ ID NO: 13, or a subsequence thereof, a sequence as set forth in SEQ ID NO: 15, or a subsequence
- the invention provides methods of amplifying a nucleic acid encoding a polypeptide having an epoxide hydrolase activity comprising amplification of a template nucleic acid with an amplification primer sequence pair capable of amplifying a nucleic acid of the invention, e.g., a nucleic acid sequence as set forth in SEQ ED NO:l, or a subsequence thereof, a sequence as set forth in SEQ ED NO:3, or a subsequence thereof, a sequence as set forth in SEQ ID NO:5, or a subsequence thereof, a sequence as set forth in SEQ ID NO:7, or a subsequence thereof, a sequence as set forth in SEQ ID NO:9, or a subsequence thereof, a sequence as set forth in SEQ ID NO: 11, or a subsequence thereof, a sequence as set forth in SEQ ID NO: 13, or a subsequence thereof, a sequence as set forth in SEQ
- the invention provides expression cassettes comprising a nucleic acid of the invention, e.g., a nucleic acid sequence having at least 50% sequence identity to SEQ ED NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ DD NO:7, SEQ ED NO:ll, SEQ ID NO:13, SEQ ED NO: 15, SEQ ED NO:17, SEQ ID NO:19, SEQ TD NO:21, SEQ ID NO:31, SEQ ID NO:33, SEQ TD NO:35, SEQ TD NO:41, SEQ TD NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:57, SEQ ED NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ED NO:67, SEQ ED NO:69, SEQ DD NO:71, SEQ DD NO:73, SEQ DD NO:75, SEQ DD NO:77, or SEQ DD NO:
- the invention provides vectors comprising a nucleic acid of the invention, e.g., a nucleic acid sequence having at least 50% sequence identity to SEQ ED NO:l, SEQ DD NO:3, SEQ DD NO:5, SEQ DD NO:7, SEQ DD NO:ll, SEQ DD NO:13, SEQ DD NO:15, SEQ DD NO:17, SEQ ID NO:19, SEQ TD NO:21, SEQ DD NO:31, SEQ DD NO:33, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ DD NO:47, SEQ DD NO:53, SEQ TD NO:57, SEQ ID NO:59, SEQ DD NO:61, SEQ DD NO:63, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ TD NO:75, SEQ ID NO:77, or SEQ ID NO:
- the invention provides cloning vehicles comprising a vector of the invention, wherein the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome.
- the viral vector can comprise an adenovirus vector, a retroviral vector or an adeno-associated viral vector.
- the cloning vehicle can comprise a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage PI -derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
- BAC bacterial artificial chromosome
- PAC bacteriophage PI -derived vector
- YAC yeast artificial chromosome
- MAC mammalian artificial chromosome
- the invention provides transformed cells comprising a vector, wherein the vector comprises a nucleic acid of the invention, e.g., a sequence having at least 50% sequence identity to SEQ ID NO:l, SEQ DD NO:3, SEQ DD NO:5, SEQ DD NO:7, SEQ DD NO:ll, SEQ DD NO:13, SEQ DD NO:15, SEQ DD NO:17, SEQ DD NO:19, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:43, SEQ TD NO:45, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:57, SEQ DD NO:59, SEQ DD NO:61, SEQ ED NO:63, SEQ D NO:67, SEQ ED NO:69, SEQ ED NO:71, SEQ ED NO:73, SEQ DD NO:75, SEQ DD NO:77, or
- the invention provides transformed cells comprising a nucleic acid of the invention, e.g., a nucleic acid sequence having at least 50% sequence identity to SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ID NO:l 1, SEQ ID NO:13, SEQ DD NO:15, SEQ DD NO:17, SEQ ED NO:19, SEQ ED NO:21, SEQ ED NO:31, SEQ ID NO:33, SEQ ED NO:35, SEQ BD NO:41, SEQ DD NO:43, SEQ ED NO:45, SEQ ED NO:47, SEQ D NO:53, SEQ JD NO:57, SEQ JD NO:59, SEQ ED NO:61, SEQ TD NO:63, SEQ ID NO:67, SEQ D NO:69, SEQ ED NO:71, SEQ ED NO:73, SEQ ED NO:75, SEQ DD NO:77, or
- the cell is a bacterial cell, a mammalian cell , a fungal cell, a yeast cell, an insect cell or a plant cell.
- the invention provides transgenic non-human animals comprising a nucleic acid of the invention, e.g., a nucleic acid sequence having at least 50%o sequence identity to SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ID NO:7, SEQ ID NO:l 1, SEQ TD NO:13, SEQ ED NO:15, SEQ ED NO:17, SEQ BD NO:19, SEQ BD NO:21, SEQ BD NO:31, SEQ BD NO:33, SEQ BD NO:35, SEQ ID NO:41, SEQ ID NO:43, SEQ ED NO:45, SEQ DD NO:47, SEQ ED NO:53, SEQ ED NO:57, SEQ ED NO:59, SEQ ED NO:61, SEQ ED NO:63, S
- the invention provides transgenic plants comprising a nucleic acid of the invention, e.g., a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:l, SEQ TD NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:ll, SEQ ID NO:13, SEQ ED NO:15, SEQ DD NO:17, SEQ DD NO:19, SEQ ED NO:21, SEQ ID NO:31, SEQ ED NO:33, SEQ ED NO:35, SEQ ED NO:41, SEQ ED NO:43, SEQ ED NO:45, SEQ ID NO:47, SEQ ED NO:53, SEQ ED NO:57, SEQ ED NO:59, SEQ ED NO:61, SEQ ED NO:63, SEQ ED NO:67, SEQ ED NO:69, SEQ ID NO:71, SEQ D NO:73, SEQ ID NO:75, SEQ ID NO:77, or SEQ ED
- the plant can be a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant or a tobacco plant.
- the invention provides transgenic seeds comprising a nucleic acid of the invention, e.g., a nucleic acid sequence having at least 50% sequence identity to SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ID NO:7, SEQ ID NO:ll, SEQ ED NO:13, SEQ DD NO:15, SEQ ID NO:17, SEQ ED NO:19, SEQ TD NO:21, SEQ TD NO:31, SEQ ED NO:33, SEQ ED NO:35, SEQ BD NO:41, SEQ ED NO:43, SEQ TD NO:45, SEQ TD NO:47, SEQ BD NO:53, SEQ ID NO:57, SEQ TD NO:59, SEQ TD NO:61, SEQ ED
- the invention provides antisense oligonucleotides comprising a nucleic acid of the invention, e.g., a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid sequence having at least 50% sequence identity to SEQ ID NO:l, SEQ TD NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:ll, SEQ ED NO:13, SEQ ED NO:15, SEQ ED NO:17, SEQ ED NO:19, SEQ DD NO:21, SEQ DD NO:31, SEQ TD NO:33, SEQ ID NO:35, SEQ ED NO:41, SEQ ED NO:43, SEQ ID NO:45, SEQ TD NO:47, SEQ ED NO:53, SEQ ID NO:57, SEQ TD NO:59, SEQ ED NO:61, SEQ BD NO:63, SEQ ED NO:67, SEQ ED NO:69, SEQ
- the invention provides methods of inhibiting the translation of an epoxide hydrolase message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid of the invention, e.g., a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid comprising a nucleic acid sequence having at least 50% sequence identity to SEQ BD NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ID NO:ll, SEQ TD NO:13, SEQ DD NO:15, SEQ TD NO:17, SEQ ID NO:19, SEQ ED NO:21, SEQ ED NO:31, SEQ TD NO:33, SEQ ED NO:35, SEQ ED NO:41, SEQ ED NO:43, SEQ TD NO:45, SEQ DD NO:47, SEQ ED NO:53, SEQ ED NO:57
- the invention provides isolated or recombinant polypeptides comprising an amino acid sequence having at least 50% identity to SEQ ED NO:2, SEQ ED NO:4, SEQ ED NO:6, SEQ ED NO:8, SEQ ED NO:12, SEQ ED NO:14, SEQ ED NO:16, SEQ ID NO: 18, SEQ DD NO:20, SEQ DD NO:22, SEQ ED NO:32, SEQ ED NO:34, SEQ ID NO:36, SEQ ED NO:42, SEQ DD NO:44, SEQ ED NO:46, SEQ BD NO:48, SEQ ED NO:54, SEQ ED NO:58, SEQ ED NO:60, SEQ TD NO:62, SEQ ED NO:64, SEQ ED NO:68, SEQ ED NO:70, SEQ ED NO:72, SEQ ED NO:74, SEQ ED NO:76, SEQ ED NO:78, or SEQ ED NO:80 over a
- the polypeptide has an epoxide hydrolase activity.
- the epoxide hydrolase activity can comprise catalyzing the addition of water to an oxirane compound.
- the epoxide hydrolase activity can further comprise formation of a corresponding diol.
- the epoxide hydrolase activity can further comprise formation of an enantiomerically enriched epoxide.
- the oxirane compound can comprise an epoxide or arene oxide.
- the oxirane compound or the corresponding diol can be optically active.
- the oxirane compound or the corresponding diol is enantiomerically pure.
- the epoxide hydrolase activity can be enantioselective.
- the epoxide hydrolase activity can comprise hydrolyzing a mono-substituted, 2,2- disubstituted, 2,3-disubstituted, trisubstituted epoxide or a styrene-oxide.
- the epoxide hydrolase activity is thermostable.
- the polypeptide can retain an epoxide hydrolase activity under conditions comprising a temperature range of between about 37°C to about 70°C.
- the epoxide hydrolase activity can be thermotolerant.
- the polypeptide can retain an epoxide hydrolase activity after exposure to a temperature in the range from greater than 37°C to about 90°C.
- the polypeptide can retain an epoxide hydrolase activity after exposure to a temperature in the range from greater than 37°C to about 50°C.
- the polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more identity to SEQ ID NO:2, SEQ ED NO:4, SEQ TD NO:6, SEQ ED NO:8, SEQ DD NO: 12, SEQ DD NO:14, SEQ ED NO:16, SEQ ED NO:18, SEQ ED NO:20, SEQ BD NO:22, SEQ ED NO:32, SEQ ED NO:34, SEQ ID NO:36, SEQ ID NO:42, SEQ ED NO:44, SEQ TD NO:46, SEQ ED NO:48, SEQ ED NO:54, SEQ TD NO:58, SEQ BD NO:60, SEQ ED NO:62, SEQ BD NO:64, SEQ ED NO:68, SEQ ID NO:70, SEQ ED NO:72, SEQ ED NO:74, SEQ ED
- the invention provides isolated or recombinant polypeptides, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ED NO:2, an amino acid sequence as set forth in SEQ ED NO:4, an amino acid sequence as set forth in SEQ ED NO:6, an amino acid sequence as set forth in SEQ ED NO:8, an amino acid sequence as set forth in SEQ ED NO:10, an amino acid sequence as set forth in SEQ ED NO:12, an amino acid sequence as set forth in SEQ ED NO: 14, an amino acid sequence as set forth in SEQ ID NO: 16, an amino acid sequence as set forth in SEQ ED NO: 18, an amino acid sequence as set forth in SEQ ED NO:20, an amino acid sequence as set forth in SEQ ED NO:22, an amino acid sequence as set forth in SEQ ED NO:24, an amino acid sequence as set forth in SEQ ED NO:26, an amino acid sequence as set forth in SEQ ED NO:28, an amino acid sequence as set forth in SEQ ED NO:30,
- the isolated or recombinant polypeptide comprising the polypeptide of the invention and lacks a signal sequence.
- the epoxide hydrolase activity comprises a specific activity at about 37°C in the range from about 100 to about 1000 units per milligram of protein. In another aspect, the epoxide hydrolase activity comprises a specific activity from about 500 to about 1200 units per milligram of protein. Alternatively, the epoxide hydrolase activity comprises a specific activity at 37°C in the range from about 500 to about 1000 units per milligram of protein. In one aspect, the epoxide hydrolase activity comprises a specific activity at 37°C in the range from about 750 to about 1000 units per milligram of protein.
- thermotolerance comprises retention of at least half of the specific activity of the epoxide hydrolase at 37°C after being heated to the elevated temperature.
- thermotolerance comprises retention of specific activity at 37°C in the range from about 500 to about 1200 units per milligram of protein after being heated to the elevated temperature.
- the invention provides the polypeptide of the invention, wherein the polypeptide comprises at least one glycosylation site.
- glycosylation can be an N-linked glycosylation.
- the epoxide hydrolase is glycosylated after being expressed in a P. pastoris or a S. pombe.
- the polypeptide can retain an epoxide hydrolase activity under conditions comprising about pH 4.5 or pH 5.
- the polypeptide can retain an epoxide hydrolase activity under conditions comprising about pH 9.0, pH 9.5, or pH lO.
- the invention provides protein preparations comprising a polypeptide of the invention, wherein the protein preparation comprises a liquid, a solid or a gel.
- the invention provides heterodimers comprising a polypeptide of the invention and a second domain.
- the second domain is a polypeptide and the heterodimer is a fusion protein.
- the second domain can be an epitope or a tag.
- the invention provides immobilized polypeptide having an epoxide hydrolase activity, wherein the polypeptide comprises a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention or a polypeptide comprising a polypeptide of the invention and a second domain.
- the polypeptide can be immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
- the invention provides arrays comprising an immobilized polypeptide, wherein the polypeptide comprises a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention or a polypeptide comprising a polypeptide of the invention and a second domain.
- the invention provides arrays comprising an immobilized nucleic acid of the invention.
- the invention provides arrays comprising an antibody of the invention.
- the invention provides isolated or recombinant antibodies that specifically binds to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
- the antibody can be a monoclonal or a polyclonal antibody.
- the invention provides hybridomas comprising an antibody that specifically binds to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
- the invention provides methods of isolating or identifying a polypeptide with epoxide hydrolase activity comprising the steps of: (a) providing an antibody of the invention; (b) providing a sample comprising polypeptides; and (c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying a polypeptide having an epoxide hydrolase activity.
- the invention provides methods of making an anti-epoxide hydrolase antibody comprising administering to a non-human animal a nucleic acid of the invention, or a polypeptide of the invention, in an amount sufficient to generate a humoral immune response, thereby making an anti-epoxide hydrolase antibody.
- the invention provides methods of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid of the invention operably linked to a promoter; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide.
- the method can further comprise transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
- the invention provides methods for identifying a polypeptide having an epoxide hydrolase activity comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing an epoxide hydrolase substrate; and (c) contacting the polypeptide or a fragment or variant thereof of step (a) with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having an epoxide hydrolase activity.
- the substrate can be an epoxide.
- the invention provides methods for identifying an epoxide hydrolase substrate comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as an epoxide hydrolase substrate.
- the invention provides methods of determining whether a test compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid comprises a nucleic acid of the invention, or, providing a polypeptide of the invention; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide.
- the mvention provides methods for identifying a modulator of an epoxide hydrolase activity comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the epoxide hydrolase, wherein a change in the epoxide hydrolase activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the epoxide hydrolase activity.
- the epoxide hydrolase activity is measured by providing an epoxide hydrolase substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increase in the amount of the substrate or a decrease in the amount of a reaction product.
- a decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of the epoxide hydrolase activity.
- An increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of the epoxide hydrolase activity.
- the invention provides computer systems comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide of the invention, or subsequence thereof, and the nucleic acid comprises a nucleic acid of the invention.
- the computer system can further comprise a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon.
- the sequence comparison algorithm comprises a computer program that indicates polymorphisms.
- the computer system can further comprise an identifier that identifies one or more features in said sequence.
- the invention provides computer readable media having stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide of the invention, or subsequence thereof, and the nucleic acid comprises a nucleic acid of the invention, or subsequence thereof.
- the invention provides methods for identifying a feature in a sequence comprising the steps of: (a) reading the sequence using a computer program which identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide of the invention or subsequence thereof, and the nucleic acid comprises a nucleic acid of the invention or subsequence thereof; and (b) identifying one or more features in the sequence with the computer program.
- the invention provides methods for comparing a first sequence to a second sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer program which compares sequences, wherein the first sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide of the invention, or subsequence thereof, and the nucleic acid comprises a nucleic acid of the invention or subsequence thereof; and (b) determining differences between the first sequence and the second sequence with the computer program.
- the step of determining differences between the first sequence and the second sequence further comprises the step of identifying polymorphisms.
- the method can further comprise an identifier that identifies one or more features in a sequence.
- the method can further comprise reading the first sequence using a computer program and identifying one or more features in the sequence.
- the mvention provides methods for isolating or recovering a nucleic acid encoding a polypeptide with an epoxide hydrolase activity from an environmental sample comprising the steps of: (a) providing an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide with an epoxide hydrolase activity, wherein the primer pair is capable of amplifying SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:9, SEQ ED NO:l l, SEQ ED NO:13, SEQ ED NO:15, SEQ ED NO:17, SEQ TD NO:19, SEQ ED NO:21, SEQ BD NO:23, SEQ ED NO:25, SEQ ED NO:27, SEQ DD NO:29, SEQ ED NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ED NO:37, SEQ ED NO:
- one and each member of the amplification primer sequence pair comprises an oligonucleotide comprising at least about 10 to 50 consecutive bases of a sequence as set forth in SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:9, SEQ ID NO:ll, SEQ ED NO:13, SEQ BD NO:15, SEQ ID NO:17, SEQ ED NO:19, SEQ ED NO:21, SEQ ED NO:23, SEQ ID NO:25, SEQ ED NO:27, SEQ DD NO:29, SEQ ED NO:31, SEQ TD NO:33, SEQ ED NO:35, SEQ ED NO:37, SEQ ED NO:39, SEQ ID NO:41, SEQ ED NO:43, SEQ ED NO:45, SEQ ED NO:47, SEQ BD NO:49, SEQ ED NO:51, SEQ ED NO:53, SEQ ID NO
- the invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide with a epoxide hydrolase activity from an environmental sample comprising the steps of: (a) providing a polynucleotide probe comprising a nucleic acid of the invention, or a subsequence thereof; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a); (c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide with an epoxide hydrolase activity from an environmental sample.
- the environmental sample comprises a water sample, a liquid sample, a soil sample, an air sample or a biological sample.
- the biological sample can be derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.
- the invention provides methods of generating a variant of a nucleic acid encoding a polypeptide with an epoxide hydrolase activity comprising the steps of: (a) providing a template nucleic acid comprising a nucleic acid of the invention; and (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid.
- the method can further comprise expressing the variant nucleic acid to generate a variant epoxide hydrolase polypeptide.
- the modifications, additions or deletions are introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof.
- a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof
- the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
- the method can be iteratively repeated until an epoxide hydrolase having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced.
- the variant epoxide hydrolase polypeptide can be thermotolerant, and retains some activity after being exposed to an elevated temperature.
- the variant epoxide hydrolase polypeptide has increased glycosylation as compared to the epoxide hydrolase encoded by a template nucleic acid.
- the variant epoxide hydrolase polypeptide has an epoxide hydrolase activity under a high temperature, wherein the epoxide hydrolase encoded by the template nucleic acid is not active under the high temperature.
- the method is iteratively repeated until an epoxide hydrolase coding sequence having an altered codon usage from that of the template nucleic acid is produced. In another aspect, the method is iteratively repeated until an epoxide hydrolase gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.
- the invention provides methods for modifying codons in a nucleic acid encoding a polypeptide with a epoxide hydrolase activity to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid encoding a polypeptide with a epoxide hydrolase activity comprising a nucleic acid of the invention; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over- represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
- the invention provides methods for modifying codons in a nucleic acid encoding an epoxide hydrolase polypeptide, the method comprising the following steps: (a) providing a nucleic acid encoding a polypeptide with an epoxide hydrolase activity comprising a nucleic acid of the invention; and, (b) identifying a codon in the nucleic acid of step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon, thereby modifying codons in a nucleic acid encoding an epoxide hydrolase.
- the mvention provides methods for modifying codons in a nucleic acid encoding a epoxide hydrolase polypeptide to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid encoding an epoxide hydrolase polypeptide comprising a nucleic acid of the invention; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
- the invention provides methods for modifying a codon in a nucleic acid encoding a polypeptide having an epoxide hydrolase activity to decrease its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid encoding an epoxide hydrolase polypeptide comprising a nucleic acid of the invention; and (b) identifying at least one preferred codon in the nucleic acid of step (a) and replacing it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in a host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell.
- the host cell can be a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or
- the invention provides methods for producing a library of nucleic acids encoding a plurality of modified epoxide hydrolase active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived from a first nucleic acid comprising a sequence encoding a first active site or a first substrate binding site the method comprising the following steps: (a) providing a first nucleic acid encoding a first active site or first substrate binding site, wherein the first nucleic acid sequence comprises a sequence that hybridizes under stringent conditions to a sequence as set forth in SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:9, SEQ ED NO:l l, SEQ ED NO:13, SEQ ED NO:15, SEQ ED NO:17, SEQ ED NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ED NO:25, SEQ ED NO:27, SEQ ED
- the method can further comprise mutagenizing the first nucleic acid of step (a) by a method comprising an optimized directed evolution system, gene site-saturation mutagenesis (GSSM), a synthetic ligation reassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, or a combination thereof.
- GSSM gene site-saturation mutagenesis
- SLR synthetic ligation reassembly
- error-prone PCR shuffling
- oligonucleotide-directed mutagenesis assembly PCR
- sexual PCR mutagenesis in vivo mutagenesis
- cassette mutagenesis cassette mutagenesis
- recursive ensemble mutagenesis recursive
- the method can further comprise mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
- the invention provides methods for making a small molecule comprising the following steps: (a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymes comprises an epoxide hydrolase enzyme encoded by a nucleic acid comprising a nucleic acid of the invention; (b) providing a substrate for at least one of the enzymes of step (a); and (c) reacting the substrate of step (b) with the enzymes under conditions that facilitate a plurality of biocatalytic reactions to generate a small molecule by a series of biocatalytic reactions.
- the invention provides methods for modifying a small molecule comprising the following steps: (a) providing an epoxide hydrolase enzyme, wherein the enzyme comprises a polypeptide of the invention, or, is encoded by a nucleic acid of the invention; (b) providing a small molecule; and (c) reacting the enzyme of step (a) with the small molecule of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the epoxide hydrolase enzyme, thereby modifying a small molecule by an epoxide hydrolase enzymatic reaction.
- the method can further comprise a plurality of small molecule substrates for the enzyme of step (a), thereby generating a library of modified small molecules produced by at least one enzymatic reaction catalyzed by the epoxide hydrolase enzyme.
- the method can further comprise a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurality of enzymatic reactions.
- the method can comprise the step of testing the library to determine if a particular modified small molecule which exhibits a desired activity is present within the library.
- the step of testing the library can comprise the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by testing the portion of the modified small molecule for the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.
- the invention provides methods for determining a functional fragment of an epoxide hydrolase enzyme comprising the steps of: (a) providing an epoxide hydrolase enzyme, wherein the enzyme comprises a polypeptide of the invention, or, is encoded by a nucleic acid of the invention; and (b) deleting a plurality of amino acid residues from the sequence of step (a) and testing the remaining subsequence for an epoxide hydrolase activity, thereby determmmg a functional fragment of an epoxide hydrolase enzyme.
- the epoxide hydrolase activity can be measured by providing an epoxide hydrolase substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product.
- the invention provides methods for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps: (a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid of the invention; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using realtime metabolic flux analysis.
- the genetic composition of the cell is modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene.
- the method can further comprise selecting a cell comprising a newly engineered phenotype.
- the method can further comprise culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.
- the invention provides methods for hydrolyzing an epoxide comprising the following steps: (a) providing a polypeptide having an epoxide hydrolase activity, wherein the polypeptide comprises a polypeptide of the invention, or, a polypeptide encoded by a nucleic acid of the invention; (b) providing a composition comprising an epoxide; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the polypeptide hydrolyzes the epoxide.
- the epoxide is mono-substituted, 2,2-disubstituted, 2,3-disubstituted, trisubstituted, or a styrene oxide.
- the invention provides methods for producing a chiral diol comprising the following steps: (a) providing a polypeptide having an epoxide hydrolase activity, wherein the polypeptide comprises a polypeptide of the invention, or, a polypeptide encoded by a nucleic acid of the invention; (b) providing a composition comprising a chiral epoxide; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the polypeptide catalyzes the conversion of the chiral epoxide to the chiral diol.
- the invention provides methods for producing a chiral epoxide comprising the following steps: (a) providing a polypeptide having an epoxide hydrolase activity, wherein the polypeptide a polypeptide of the invention, or, a polypeptide encoded by a nucleic acid of the invention, wherein the epoxide hydrolase activity is enantioselective or enantiospecific; (b) providing a composition comprising a racemic mixture of chiral epoxides; (c) combining the polypeptide of step (a) with the composition of step (b) under conditions wherein the enantioselective or enantiospecific polypeptide converts the epoxide substrate of the specific chirality to a diol, thereby leading to accumulation of the unreacted epoxide of the opposite chirality.
- the invention provides methods of increasing thermotolerance or thermostability of an epoxide hydrolase polypeptide, the method comprising glycosylating an epoxide hydrolase polypeptide, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, thereby increasing the thermotolerance or thermostability of the epoxide hydrolase polypeptide.
- the epoxide hydrolase specific activity is thermostable or thermotolerant at a temperature in the range from greater than about 37°C to about 90°C.
- the invention provides methods for overexpressing a recombinant epoxide hydrolase polypeptide in a cell comprising expressing a vector comprising a nucleic acid comprising a nucleic acid sequence at least 50% sequence identity to a nucleic acid of the invention over a region of at least about 100 residues, wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection, wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.
- the invention provides growth-based methods for selecting a cell comprising a nucleic acid encoding an epoxide hydrolase comprising the following steps:
- step (a) providing a plurality of cells, wherein the cells lack a composition essential for growth; (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by an epoxide hydrolase to a composition essential for growth of the cells; (c) growing the cells in a medium lacking a carbon source essential for growth and adding the precursor or substrate of step (b); and, (d) screening the cells for growth, wherein the cells in the growth stimulated clone are identified as comprising the nucleic acid encoding an epoxide hydrolase capable of converting the precursor or substrate to the composition essential for growth, thereby selecting a cell comprising a nucleic acid encoding an epoxide hydrolase.
- the invention provides growth-based methods for selecting a nucleic acid encoding an epoxide hydrolase comprising the following steps: (a) providing a nucleic acid encoding a polypeptide; (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by an epoxide hydrolase to a composition essential for growth of the cell; (c) providing a plurality of cells, wherein the cells cannot make the composition of step (b); (d) inserting the nucleic acid into the cells and growing the cells under conditions wherein the nucleic acid is expressed and its encoded polypeptide is translated, and the cells are grown in a medium lacking the carbon source essential for growth, and adding the precursor or substrate of step (b); and, (e) screening the cells for growth, wherein the nucleic acid in the growth stimulated clone is identified as encoding an epoxide hydrolase capable of converting the precursor or substrate to the composition comprising essential for growth, thereby selecting a nucleic acid encoding an
- the invention provides methods for identifying a nucleic acid encoding an epoxide hydrolase comprising the following steps: (a) providing a nucleic acid library;
- step (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by an epoxide hydrolase to a composition essential for growth of the cells; (c) providing a plurality of cells, wherein the cells cannot make the composition of step (b); (d) inserting in a cell a member of the gene library and culturing the cells in a medium lacking the composition essential for growth; (e) adding the precursor or substrate of step (b) to the culture; (f) selecting a growing cell and identifying the inserted library member of step (d), wherein the cell is capable of growth by enzymatic conversion of the precursor to the composition essential for growth, and the enzyme is encoded by the library member, thereby identifying a nucleic acid encoding an epoxide hydrolase.
- the precursor or substrate comprises glycidol or propylene oxide.
- the composition essential for growth comprises glycerol or propane diol.
- the precursor or substrate comprises a pure enantiomer or a racemic mixture.
- the composition essential for growth can comprise a pure enantiomer or a racemic mixture.
- the nucleic acid is a member of a gene library.
- the library can be obtained from a mixed population of organisms. The mixed population of organisms is derived from a soil sample, a water sample or an air sample.
- the cells comprise E. colifucA-disrv ⁇ ted mutant.
- the invention provides methods for identifying an epoxide hydrolase comprising the following steps: (a) providing a polypeptide; (b) providing a precursor or substrate, wherem the precursor or substrate is capable of being converted by an epoxide hydrolase to a composition essential for growth of the cells; (c) providing a plurality of cells, wherein the cells cannot make the composition of step (b); (d) inserting the polypeptide into the cells and culturing the cells, and the cells are grown in a medium lacking the composition essential for growth, and adding the precursor or substrate of step (b); and, (e) screening the cells for growth, wherein the polypeptide in the growth stimulated clone is identified as being an epoxide hydrolase capable of converting the precursor or substrate to a composition essential for growth of the cells, thereby identifying an epoxide hydrolase.
- the invention provides methods for identifying an epoxide hydrolase comprising the following steps: (a) providing a polypeptide library; (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by an epoxide hydrolase to a composition essential for growth of the cells; (c) providing a plurality of cells, wherein the cells cannot make the composition of step (b); (d) inserting in a cell a member of the polypeptide library and culturing the cells in a medium lacking the composition essential for growth; (d) adding the polypeptide library of step (a) and the precursor or substrate of step (b) to the cells of step (c); and (f) selecting a growing cell and identifying the inserted polypeptide of step (d), wherein the cell is capable of growth by enzymatic conversion of the precursor to the composition essential for growth, thereby identifying an epoxide hydrolase.
- the library is obtained from a mixed population of organisms.
- the invention provides direct activity assay methods for screening for a polypeptide having an epoxide hydrolase activity comprising the following steps: (a) providing a plurality of polypeptides; (b) providing a precursor or substrate covalently linked to a fluorophore, wherein the precursor or substrate is capable of being converted by an epoxide hydrolase to a diol, wherein the fluorophore can generate a fluorescent signal when free; (c) combining the polypeptides of step (a) with the precursor or substrate of step (b) under conditions wherein the polypeptides can convert the precursor or substrate to a diol linked to the fluorophore; (d) converting the diol linked to the fluorophore of step (c) to a free fluorophore; (e) measuring the fluorescence quantum yield; and (f) screening the polypeptides for epoxide hydrolase activity, wherein the polypeptide is identified as having an epoxide hydrolase activity capable of converting the
- the conversion of the diol linked to the fluorophore to free fluorophore further comprises the following steps: (a) subjecting the diol linked to the fluorophore of step to periodate oxidation resulting in the formation of an aldehyde linked to the fluorophore; (b) subjecting the aldehyde of step (a) to a BSA-catalyzed ⁇ -elimination resulting in the formation of the free fluorophore.
- the fluorophore can be umbellipherone.
- the invention provides direct activity colorimetric methods for screening for a polypeptide having an epoxide hydrolase activity comprising the following steps: (a) providing a plurality of polypeptides; (b) providing a precursor or substrate, wherein the precursor or substrate is capable of being converted by an epoxide hydrolase to a diol, (c) providing a chemical, wherein the chemical is capable of reaction with the precursor or substrate forming a product capable of absorbance at a visible wavelength, wherein the chemical is not reactive with the diol; (d) combimng the polypeptide of step (a) with the precursor or substrate of step (b) under conditions wherein the polypeptide can convert the precursor or substrate to the diol; (e) measuring a decrease of light absorbance at the wavelength characteristic for absorbance of the product linked to the precursor or substrate; and (f) screening the polypeptides for epoxide hydrolase activity, wherein the polypeptide is identified as having an epoxide hydrolase activity capable of converting the precursor or substrate to
- the invention provides in vitro growth selection screens using epoxides as precursors to discover nucleic acids encoding epoxide hydrolases that produce a diol product comprising the following steps: (a) providing a nucleic acid library; (b) providing a precursor, wherein the precursor is capable of being converted to a diol; (c) providing an in vitro transcription/ translation system lacking the diol; (d) adding to the in vitro transcription/ translation system a member of the nucleic acid library; (e) adding the precursor of step (b); and (f) selecting a sample producing the diol and identifying the inserted nucleic acid of step (d), wherein selecting the sample comprising the precursor selects a nucleic acid encoding a corresponding epoxide hydrolase.
- the invention provides in vitro growth selection screens using epoxides as precursors to discover epoxide hydrolases that produce a diol comprising the following steps: (a) providing a polypeptide library; (b) providing a precursor, wherein the precursor is capable of being converted to a diol; (c) providing an in vitro transcription/ translation system lacking the diol; (d) adding to the in vitro transcription/ translation system a member of the polypeptide library; (e) adding the precursor of step (b); and (f) selecting a sample producing the diol and identifying the added polypeptide of step (d), wherein selecting the sample comprising the diol selects a corresponding epoxide hydrolase.
- Figure 1 is a schematic representation of the selective hydrolysis of a racemic epoxide generating the corresponding diol and the unreacted epoxides with high enantiomeric excess (ee) values.
- Figure 2 is a schematic representation of glycidols, (S-(l), and R-(2)), the leading chiral epoxides among representative C-3 synthons.
- Figure 3 is a schematic representation of the production of saquinavir, an antiviral drug and the synthesis of amprenavir, another antiviral drug.
- Figure 4 is a schematic representation of the synthesis of two anticancer drugs, docetaxel and paclitaxel.
- Figure 5 is a schematic representation of hydrolysis of styrene-oxide types of substrates by A. niger epoxide hydrolase hydrolyzing R-enantiomers in all transformations.
- Figure 6 is a chart summary of exemplary reactions that can be used with the epoxide hydrolases of the invention.
- Figure 7 is a schematic representation of an exemplary reaction where an epoxide hydrolase of the invention is used in the desymmetrization of meso-epoxides.
- Figure 8 is a block diagram of a computer system.
- Figure 9 is a flow diagram illustrating one aspect of a process for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
- Figure 10 is a flow diagram illustrating one aspect of a process in a computer for determining whether two sequences are homologous.
- Figure 11 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence.
- Figure 12 is an illustration of the mechanism of A. radiobacter epoxide hydrolase.
- Figure 13 is an illustration of the types of epoxide substrates.
- Figure 14 is an illustration of the enantioconvergent hydrolysis of cis-2,3 - epoxyheptane to 2R,3R-2,3-dihydroxyheptane catalyzed by Norcardia EH1.
- Figure 15 is an illustration of glycidol and propylene oxide used as selection substrates.
- Figure 16 is an illustration of a high-throughput screening method based on a periodate-coupled fluorogenic assay for an epoxide hydrolase.
- Figure 17 is an illustration of the synthesis of the substrates for a periodate- coupled fluorogenic assay for an epoxide hydrolase.
- FIG. 18 is an illustration of Fluorescence Activated Cell Sorting (FACS) for ultra high throughput single cell activity and sequence screening.
- Figure 19 is an illustration of environmental library biopamiing for sequence- based discovery.
- the invention provides polypeptides having epoxide hydrolase activity, polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides.
- the polypeptides of the invention can be used as epoxide hydrolases to catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols.
- Epoxide hydrolases of the mvention can be hydrolytic enzymes to catalyze the opening of an epoxide ring to convert a substrate to a corresponding diol.
- Epoxide hydrolases of the invention can be highly regio- and enantioselective, allowing the preparation of pure enantiomers.
- the polypeptides of the invention can be used to hydrolyze hazardous epoxide compounds generated through peroxidation in living organisms, and, to eliminate the high chemical reactivity of epoxide compounds.
- the invention provides epoxide hydrolases (EHs) from wide varieties of biodiversity sources such as enzyme or gene libraries.
- the invention provides methods to rapidly select or screen enzymes and genes to obtain suitable EHs.
- the invention provides methods to access untapped biodiversity and to rapidly screen for sequences and activities of interest utilizing recombinant DNA technology. This invention combines the benefits associated with the ability to rapidly screen natural compounds with the flexibility and reproducibility afforded with working with the genetic material of organisms.
- the invention provides method to synthesize useful chiral epoxides using the enzymes of the present invention.
- the invention provides useful chiral epoxides and their derivatives produced using the EHs of the present invention.
- the epoxide hydrolases of the invention are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolyzed epoxide in nonracemic form, the epoxide hydrolases of the mvention are used in enantioconvergent processes for the generation of a single enantiomeric diol from a racemic oxirane.
- the epoxide hydrolases of the invention can be used in the hydrolysis of highly substituted epoxides, e.g., highly substituted 2,2- and 2,3-disubstituted epoxides.
- the epoxide hydrolases of the invention can be used in any method known in the art, see, e.g., Orru (1999) Curr. Opin. Chem. Biol. 3:16-21.
- polypeptides of the invention can be used as epoxide hydrolases in Sharpless epoxidation, Katsuki- Jacobsen reactions, Shi Epoxidation and Jacobsen hydrolytic kinetic resolution reactions (see Figure 6).
- the invention provides methods for using epoxide hydrolases of the invention to provide stereospecific reaction products.
- the polypeptides of the invention can be used in the desymmetrization of meso-epoxides.
- the conversion of substrate to either R,R or S,S-product was with greater than 97%ee, and, in one aspect, 99% conversion.
- Figure 7 is a schematic of an exemplary reaction where an epoxide hydrolase of the invention is used in the desymmetrization of meso-epoxides.
- the invention provides epoxide hydrolases to produce styrene glycol, and corresponding methods. The epoxide hydrolases are reacted with styrene oxide to produce styrene glycols.
- the invention provides methods for enzymatic separation of epoxide- enantiomer mixtures.
- the invention provides methods for protecting a cell against oxidants, e.g., in an immunotoxic reaction, comprising introducing around or into the cell an antioxidizing agent comprising an epoxide hydrolase.
- the invention provides epoxide hydrolase inhibitors (e.g., an antisense or ribozyme nucleic acid, or an antibody, of the invention) to ameliorate an immunological disorder, e.g., a T cell mediated disorder, and corresponding methods of ameliorating an immunological disorder, e.g., a T cell mediated disorder.
- the invention provides epoxide hydrolases to treat peroxisomal disorders, and corresponding methods of ameliorating a peroxisomal disorder.
- the invention provides epoxide hydrolases to treat dysfunction, damage or diseases of the respiratory system and corresponding methods of ameliorating dysfunction, damage or diseases of the respiratory system.
- the invention provides reagents for forensic analyses, e.g., as chromosome markers or tissue or organ specific markers, comprising epoxide hydrolases of the invention.
- the invention provides epoxide hydrolases to develop novel pest control, e.g., insect, agents, and, compositions comprising epoxide hydrolase inhibitors (e.g., an antisense or ribozyme nucleic acid, or an antibody, of the invention) for use in pest control.
- epoxide hydrolase inhibitors e.g., an antisense or ribozyme nucleic acid, or an antibody, of the invention
- the invention provides epoxide hydrolases to hydrolyze leukotrienes, and corresponding methods, e.g., their use as anti-inflammatory reagents.
- the invention provides pharmaceutical compositions comprising one or more epoxide hydrolases of the invention to act as anti-inflammatory reagents by hydrolyzing leukotrienes and other inflammation-causing compositions.
- inflammation can be treated by inhibition of epoxide hydrolases using compositions comprising epoxide hydrolase inhibitors (e.g., an antisense or ribozyme nucleic acid, or an antibody, of the invention) to inhibit inflammation mediates by poly-unsaturated lipid metabolites.
- the invention provides epoxide hydrolases and methods to evaluate the cytotoxicity of a compound by measuring the expression of epoxide hydrolase in a cell.
- polypeptides of the invention can be made or used as epoxide hydrolases in any known method, protocol or industrial use, as described, e.g., in U.S. Patent Nos. 6,387,668; 6,379,938; 6,372,469; 6,372,469; 5,635,369; 6,174,695, describing use of epoxide hydrolase inhibitors to inhibit inflammation mediated by poly-unsaturated lipid metabolites; 5,759,765, describing epoxide hydrolases and methods to evaluate the cytotoxicity of a compound by measuring the expression of epoxide hydrolase in a cell; and, WO 01/46476, describing use of epoxide hydrolases to provide stereospecific reaction products; WO 01/07623, WO 00/68394, WO 00/37619, describing methods for enzymatic separation of epoxide-enantiomer mixtures; WO 99/06059, describing a method for
- Patent No. 6,153,397, 6,143,542, 6,037,160, and WO 99/32153 describing use of epoxide hydrolase inhibitors in pest control
- JP 20217597 describing use of epoxide hydrolases to produce styrene glycol by reaction with styrene oxides
- WO 00/50577 describing the use of epoxide hydrolases to hydrolyze leukotrienes and to act as anti-inflammatory reagents.
- epoxide hydrolase encompasses enzymes catalyzing the cofactor independent hydrolysis of oxirane compounds, for example, epoxides, to their corresponding diols by addition of a water molecule.
- the term also includes epoxide hydrolases capable of hydrolyzing peptide bonds at high temperatures, low temperatures, alkaline pHs and at acidic pHs.
- An epoxide hydrolase activity includes an epoxide hydrolase regioselective activity, i.e., when two possible carbons of the substrate are attacked.
- An epoxide hydrolase activity also comprises an enantioselective epoxide hydrolase activity, i.e., a preference of the enzyme for the substrates of certain chirality.
- An epoxide hydrolase activity comprises an epoxide hydrolase activity, which is not stereoselective.
- An "epoxide hydrolase variant” has an amino acid sequence wliich is derived from the amino acid sequence of a "precursor epoxide hydrolase".
- the precursor epoxide hydrolases include naturally-occurring epoxide hydrolases and recombinant epoxide hydrolases.
- the amino acid sequence of the epoxide hydrolase variant is "derived” from the precursor epoxide hydrolase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence.
- Such modification is of the "precursor DNA sequence" which encodes the amino acid sequence of the precursor epoxide hydrolase rather than manipulation of the precursor epoxide hydrolase enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein, as well as methods known to those skilled in the art
- antibody includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yannush (1992) J. Biochem. Biophys. Methods 25:85-97.
- antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
- Antigen binding sites e.g., fragments, subs
- array or “microarray” or “biochip” or “chip” as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface, as discussed in further detail, below.
- target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface, as discussed in further detail, below.
- computer computer program
- processor are used in their broadest general contexts and incorporate all such devices, as described in detail, below.
- expression cassette refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as an epoxide hydrolase polypeptide of the invention) in a host compatible with such sequences.
- Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers.
- “Operably linked” as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.
- expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA” vector, and the like.
- a “vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
- the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
- Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
- Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Patent No. 5,217,879), and includes both the expression and non- expression plasmids.
- a recombinant microorganism or cell culture is described as hosting an "expression vector” this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s).
- the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
- “Plasmids” can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
- gene means a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability.
- Genes can include, inter alia, regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
- nucleic acid or “nucleic acid sequence” as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single- stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs).
- DNA or RNA e.g., mRNA, rRNA, tRNA
- PNA peptide nucleic acid
- DNA-like or RNA-like material natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs
- nucleic acids i.e., oligonucleotides, containing known analogues of natural nucleotides.
- the term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Straussense Nucleic Acid Drug Dev 6:153- 156.
- amino acid or “amino acid sequence” as used herein refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.
- polypeptide and “protein” as used herein, refer to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids.
- polypeptide also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides.
- the peptides and polypeptides of the mvention also include all “mimetic” and “peptidomimetic” forms, as described in further detail, below.
- isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
- a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
- Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
- an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
- Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
- the mvention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.
- the term "recombinant" means that the nucleic acid is adj acent to a "backbone" nucleic acid to wliich it is not adjacent in its natural environment.
- nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules.”
- "Backbone molecules" include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest, h one aspect, the enriched nucleic acids represent 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules.
- Recombinant polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein.
- synthetic polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below. [0065] A promoter sequence is "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
- Oligonucleotide refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. [0067] "Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing.
- Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations.
- Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art.
- stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below.
- nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein.
- variants refers to polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of an epoxide hydrolase of the invention.
- Variants can be produced by any number of means included methods such as, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof.
- Techmques for producing variant epoxide hydrolase having activity at a pH or temperature for example, that is different from a wild-type epoxide hydrolase, are included herein.
- GSSM saturation mutagenesis
- optical directed evolution system or “optimized directed evolution” includes a method for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below.
- synthetic ligation reassembly or “SLR” includes a method of ligating oligonucleotide fragments in a non-stochastic fashion, and explained in detail, below.
- the invention provides nucleic acids, including expression cassettes such as expression vectors, encoding the polypeptides of the invention.
- the invention also includes methods for discovering new epoxide hydrolase sequences using the nucleic acids of the invention. Also provided are methods for modifying the nucleic acids of the invention by, e.g., synthetic ligation reassembly, optimized directed evolution system and/or saturation mutagenesis.
- nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.
- homologous genes can be modified by manipulating a template nucleic acid, as described herein.
- the invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.
- RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
- these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Terra. Lett. 22:1859; U.S. Patent No. 4,458,066.
- nucleic acids such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
- Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
- Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet.
- MACs mammalian artificial chromosomes
- a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.
- the invention provides fusion proteins and nucleic acids encoding them.
- a polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
- Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like.
- Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
- metal chelating peptides such as polyhistidine tracts and histidine- tryptophan modules that allow purification on immobilized metals
- protein A domains that allow purification on immobilized immunoglobulin
- the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle WA.
- the inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification.
- an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-414).
- the histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
- Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
- the invention provides nucleic acid (e.g., DNA) sequences of the invention operatively linked to expression (e.g., transcriptional or translational) control sequence(s), e.g., promoters or enhancers, to direct or modulate RNA synthesis/ expression.
- expression control sequence can be in an expression vector.
- Exemplary bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp.
- Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I.
- Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lad promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter.
- PGK 3-phosphoglycerate kinase
- Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late S V40 promoter, LTRs from retroviruses, and the mouse metallothionein-I promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses may also be used. Expression vectors and cloning vehicles
- the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the proteins of the invention.
- Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), PI -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast).
- Vectors of the invention can include chromosomal, non- chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Exemplary vectors are include: bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda- ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTl, ⁇ SG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector may be used so long as they are replicable and viable in the host. Low copy number or high copy number vectors may be employed with the present invention.
- the expression vector may comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator.
- the vector may also include appropriate sequences for amplifying expression.
- Mammalian expression vectors can comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
- DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
- the expression vectors contain one or more selectable marker genes to permit selection of host cells containing the vector.
- selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRPl gene.
- Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers.
- CAT chloramphenicol transferase
- Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells may also contain enhancers to increase expression levels.
- Enhancers are cw-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers.
- a DNA sequence may be inserted into a vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases.
- the vector may be in the form of a plasmid, a viral particle, or a phage.
- vectors include chromosomal, non-chromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
- cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by, e.g., Sambrook.
- Particular bacterial vectors which may be used include the commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA) ⁇ QE70, pQE60, ⁇ QE-9 (Qiagen), pDIO, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8 and pCM7.
- eukaryotic vectors include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
- any other vector may be used as long as it is replicable and viable in the host cell.
- Host cells and transformed cells include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
- any other vector may be used as long as it is replicable and viable in the host cell.
- Host cells and transformed cells include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
- any other vector may be used as long as it is replicable and viable in the host cell.
- the invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding a polypeptide of the invention, or a vector of the invention.
- the host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
- Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
- Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
- Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line.
- the selection of an appropriate host is within the abilities of those skilled in the art.
- the vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
- the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention.
- the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
- Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
- Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
- the expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
- Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.
- HPLC high performance liquid chromatography
- Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.
- the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
- polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated.
- Polypeptides of the invention may or may not also include an initial methionine amino acid residue.
- Cell-free translation systems can also be employed to produce a polypeptide of the invention.
- Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof.
- the DNA construct may be linearized prior to conducting an in vitro transcription reaction.
- the transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
- the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
- selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
- nucleic acids encoding the polypeptides of the invention, or modified nucleic acids can be reproduced by, e.g., amplification.
- the invention provides amplification primer sequence pairs for amplifying nucleic acids encoding epoxide hydrolase polypeptides, where the primer pairs are capable of amplifying nucleic acid sequences including the exemplary S ⁇ Q ⁇ D NO:l, or a subsequence thereof; a sequence as set forth in S ⁇ Q ⁇ D NO:3, or a subsequence thereof; a sequence as set forth in S ⁇ Q ⁇ D NO:5, or a subsequence thereof; and, a sequence as set forth in S ⁇ Q ⁇ D NO: 7, or a subsequence thereof, a sequence as set forth in S ⁇ Q ID NO: 9, or a subsequence thereof.
- One of skill in the art can design amplification primer sequence pairs for any part of or the full length of these sequences; for example:
- the exemplary S ⁇ Q BD NO:l is atgtcaaaca acgctcccca atcctcgtcg cgccgccatt tcgtcggcgt ggccgctgcg 60 gcgctcgcga caggctcgct gagccggctc gcctttgcca acgcattccc gactgtcggc 120 acgatcacgg aacccgccaa tggcgacaag gcagcgctgc gcccgttccg cgttcacatt 180 cctgaagcgc agctcgtcga catgcggcgg cgcatcaagg cgacgcgctg gcggaccgc 240 gaaaccgtgc ccgacgaatc gcagggtatt ca
- an exemplary amplification primer sequence pair is residues 1 to 21 of SEQ ED NO:l and the complementary strand of the last 21 residues of SEQ ED NO:l.
- the exemplary SEQ ED NO: 1 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:3 is atgcgggtgc agctgtccga ggtgaacctc gacgtcgagg tgagcgggga ggggccggcc 60 gtgctgctcg tgcacggctt ccccgacagc catcgtctgt ggcgtcatca ggtcgcggcg 120 ctgaacgacg ccggtttcac cacggtcgcg cccaccctgc ggggcttcgg cgcctcggac 180 cgccccgagg gcggccccgc ggcgtaccac ccgggcaggc acgtcgccgagtggtcgag 240 ctcctggcgc acctcgacctacct
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO: 3 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:5 is atgaggccaa cctccacacc cgagggcccc ggctccgtct ccggggcacc caacctcccg 60 gaggggttcg ccgacacctt caccagcagg tacgtcgacg ccggtgagct gcgtctccat 120 gcagttaccg gcggcgaagg cccgcccctg ctc acgggtggcc cgacgggtggcc cgagacctgg 180 tacgcctggc ggatggtgat gccggcgttg gccgagcact tcgaggtgat cgcggtcgac 240 cagcgcgggg tcgggctgtc cgacaagcccc gaggacgg
- the exemplary SEQ ED NO:5 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:7 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:9 is atgagtgtcg ttacagaaca cactgacaag accgctattc gtccgttcaa gatcaatgtg 60 ccggaggcgg acctgaagga tttgcacagg cgcatccagg cgaccaagtt tcccgaacgc 120 gagacggttc cggatgccac gcagggcgtg cagcttgcca cggttcaggc cctcgcgcag 180 tattgggcga agactacaa ctggcacaag tgtgagtcga ggctgaatgc actgccgcag 240 ttcatgaccg agattgaggg gctcgacatt catttcattc acgt
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO: 9 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:ll is atgagcaaca cacacgtcgc cgccgggacg gagatccgcc ccttcaccgt cgaggtcgcc 60 caagacgagt tggacgacct cagccgtcgc atctcggcga cgcgctggcc cgaggaggag 120 accgtcgagg atcagtcgca gggcgtgccg ctggcgacga tgcaggagct cgctac 180 tggggctccg agtacgactt cggaaggctg gaggcacggt tgaacgcctt c 240 atcaccgaga tcgacggcct cgacatccac t
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO: 11 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO: 13 is atgatttcgc tcttcgccc cggaatcctc gccatcgcgc tcggcagcgc gcaggcgccg 60 cgcgacgatg tgttcgatcg cgtgacgcac ggttacgcga cgtcggatgg cggcgtgaag 120 atccactacg cgtcgctcgg ccaggggccg ctcgtggtga tgatccacgg cttccggat 180 ttctggtact cgtggcggcg ccagatgcaa gcgttgtcgg atcgctatca ggtggtcgcc 240 atcgatcagc gcggctacaa cctgagcgacgctgag
- an exemplary amplification primer sequence pair is residues 1 to 21 of SEQ ID NO:13 and the complementary strand of the last 21 residues of SEQ ID NO:13.
- the exemplary SEQ ED NO: 13 encodes a polypeptide having the sequence
- the exemplary SEQ BD NO: 15 is gtgagagcag gtagggttcg ggcgcggg atcgagttcg cgacgctgga ggagggcaac 60 ggtccgctcg tcctctgcct gcacgggttc cccgatcatc cccgctcgtt ccggcaccag 120 ctgccggcgc tcgcgaaggc cggattccgc gcggtcgcgccgcgcgcgctccg tggctacgcg 180 ccgaccgggc cggccccga cggccctat cagtcggcgg cgg cgcgccat ggatgccgtc 240 gcgctgatcg aggcactcgg
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- SEQ ID NO:15 and the complementary strand of the last 21 residues of SEQ BD NO:15.
- the exemplary SEQ BD NO: 15 encodes a polypeptide having the sequence
- the exemplary SEQ BD NO: 17 is atggcgaggg tcaatcgacg gttgacggtt ttcggactcg tagtcgcgct gtcggtcgtg 60 ggcgcacggg cggctcagac ccagcgtgcg tcgaactcct tcgctgcagg cgcgggcgcg 120 aagactgcct caggcgaagc gatcgtgcct tcaagatcc atgttcccga ctctgtcgtg 180 gccgacctga agcagcggct ccagcgcgcc cggttgcgg acgagattcc cgaggtggga 240 tgggactatg gcacgaacct ggcctatcg
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- SEQ ED NO:17 and the complementary strand of the last 21 residues of SEQ ED NO:17.
- the exemplary SEQ ED NO: 17 encodes a polypeptide having the sequence
- the exemplary SEQ BD NO: 19 is atgagcgaag taaacatcg cgaggtagat acgaacggta tccgcatgca catcgctgaa 60 agcgggacgg gcccgttggt gttgctgtgc catggtttc ccgaatcttg gtattcgtgg 120 cgccaccagt tggatgcggt cgcagaagct ggattccacg tggttgcacc tgacatgcga 180 ggttatggcc taactgagag tccagaagaa atcgaccggt acaccctcct ccattggtc 240 ggggatatgg tcggctgct ggacgctct ggggaggagaggaga gggggtc 240
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO: 19 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:21 is gtgagagtag aggcagacgg cgtcgggatc tcgtacgagg tgaccggaca gggacggccg 60 gtgatcctgc tgcacggctt cccagactcg ggacggcttt ggcgcaacca ggtgccggct 120 ttggctgagg ccggcttcca ggtgatcgtc cctgacctgc gcgggtacgg gcagtccgat 180 aagccagagg ccgtcgatgc gtactcccttt ccgg cggggacgt catggcggta 240 ctggctgatg cgggcgtcga tggcccac gtcgtgggccccgtggggacgt
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ID NO:21 encodes a polypeptide having the sequence
- the exemplary SEQ ID NO:23 is atgaccccga ccgttgcgac aaaaccagc gaccagcaga cagcggagaa gacagcgatt 60 cggccgtttc gcatcaacgt tcccgacgcg gaactgaccg acctgcgcag gcgcgtcagc 120 gcgacgaggt ggcccgaacg cgagacggtt ccggatcaaa cgcagggcgt gcagctcgcg 180 acggttcaac agcttgcgcg ttattgggcg accgagtacg actggcgtaa gtgcgaggcg 240 aggctgaatg ccctgcgca gttcatcacg gag
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO:23 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:25 is atgtccgaac cctggaagca tcacgccaaa gttgtcaacg gctttcgtat gcactatgtc 60 attgccggtt ccggctaccc actcgtattt ctgcatggct ggccccagag ttggtatgag 120 tggcgaaaga tcattccggc actcgctgag aagttcacgg taattgcccc ggacctacgc 180 ggattgggag attctgaacg tcctctcaca gggtatgata aacgtaccct ggcctcagat 240 gtgtacgagt tggtgaaatc ctttc ag
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO:25 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:27 is atgacacgcg actcactcca actcgccgcc gtcgcgttgg ccatggtgct cgccggcgcc 60 ttcgcgattc ccgggtgggcggcaaaccacc gtcggcagcg atgcctcgat ccgtccttc 120 aagatccaag tgccgcaagc ctcgctcgac gacctgcgcc ggcgtattgc ggcaacgcgc 180 tggcccgaca aggagaccgt cgacaacgca tcccagggcg cgcagcttgc gcagatgcag 240 gagctcgtga ggtactgggg cacgagctac gactggcgca
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO:27 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:29 is atgcatgaga taaagcatcg cgttgtcgaa acgaatggca tccgcatgca cgtcgctgag 60 tgcggggtgg gtccgcttgt gctcctgtgt cacgggtttc ccgagtgttg gtattcgtgg 120 cgccatcagt tgccggcct cgcggaagct ggattccacg tcgtcgcc tgacatgcga 180 ggctacggcg agacagaccg gccacaggaa atcgaggagt acacgctcct gcatttagtt 240 ggtgacatga taggtctgct cgacgt
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- SEQ ED NO:29 and the complementary strand of the last 21 residues of SEQ ED NO:29.
- the exemplary SEQ BD NO:29 encodes a polypeptide having the sequence
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO:31 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:33 is atgcagctcg aaaaagcgca gtacatgccc gccttagcgt catcgcacac ttggcgcagc 60 tttcttcgct acataacagt cgcgtgcttt ttgggcattt tcctgctcgg cgctcagagc 120 tacgcccaga ccggtaggac cgccatcgcg gaggcctccg tcagcagctcctgcg ISO aagccgcctg cagcgaccga agataggcg atccgtccttt tcgcgtcca cgtcccacaa 240 gaggcgctcg acgacctcag ccgtcgcgcgtcccacaa 240
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- SEQ ED NO:33 and the complementary strand of the last 21 residues of SEQ ED NO:33.
- the exemplary SEQ ED NO:33 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:35 is atgaacttca ataccgtcga ggtcacaggc cttaagatct tctaccgcga ggccgggaac 60 ccgtcaaagc cggccatcgt cctgctgcac gggttccctt cgtcctcgta ctcattccac 120 gatctcattc cgctcctgtc ggatcgtttt catgtcattg cgccggacta ccccggcatg 180 gggtacagcg aagcgccacc cacgggcgca atgcgcccga ctttcgacga tatggtgaag 240 gccatggaca catttatcgc ccaatgtgcc ctgggc
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- SEQ ED NO:35 and the complementary strand of the last 21 residues of SEQ ED NO:35.
- the exemplary SEQ ED NO:35 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:37 is atgacccaga cgacaacccg ccctgccatc cgctccttcg aggtctcctt tcccgatgaa 60 gcactcgcgg acctccgccg gcgcttagca gcgacgcgct ggccggagaa agagaccgtc 120 gccgacaact cacaaggcgt cccgctggtc aacatgcagc agctggcccg ctactgggcg 180 gccgaatacg actggcgcaa gacggaggcg aagctcaacg ccttgcccca attcctgact 240 gaaatcgacg ggctgggcat tcacttcatt cactgtgact 240 gaaatcg
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO:37 encodes a polypeptide having the sequence
- the exemplary SEQ ED NO:39 is atgacctcag agaaactgca gtacccggcg agaactcaaa cgacccgcct tagcgccgcc 60 gcggcggccg ggcttgcctc gggacttctc gtcttcttt gcccgaatta cggccagacc 120 accaccgatc gtgggagcgc gatcgtcgcc caggcgtctg cgcagcgcggcagcggaa 180 gatccatcga tccgcccctt caaggtgcaa ataccgcaag ccgctcga cgacctgcgc 240 cggcgcatca acgccacgcg ctggccgac aggagaccgaccgcgc 240 cggcgcatca a
- an exemplary amplification primer sequence pair is residues 1 to 21 of
- the exemplary SEQ ED NO:39 encodes apolypeptide having the sequence
- the invention provides nucleic acids and polypeptides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% sequence identity (homology) to SEQ ED NO:l, SEQ ED NO:2, SEQ ED NO:3, SEQ BD NO:4, SEQ BD NO:5, SEQ ED NO:6, SEQ ID NO:7, SEQ ED NO:8, SEQ ED NO:9, SEQ ED NO: 10, SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, SEQ ID NO:14, SEQ ED NO:15, SEQ ED NO:16, SEQ BD NO:17, SEQ ED NO:18, SEQ ED NO:19, SEQ BD NO:20.
- sequence identify can be over a region of at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, consecutive residues, or the full length of the nucleic acid or polypeptide.
- the extent of sequence identity may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.
- Homologous sequences also include RNA sequences in which uridines replace the thymines in the nucleic acid sequences.
- the homologous sequences may be obtained using any of the procedures described herein or may result from the correction of a sequencing error. It will be appreciated that the nucleic acid sequences as set forth herein can be represented in the traditional single character format (see, e.g., Stryer, Lubert. Biochemistry, 3rd Ed., W H Freeman & Co., New York) or in any other format which records the identity of the nucleotides in a sequence. [00160] Various sequence comparison programs identified herein are used in this aspect of the invention. Protein and/or nucleic acid sequence identities (homologies) may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
- Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403- 410, 1990; Thompson et al, Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).
- homology or identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications.
- sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705
- one sequence can act as a reference sequence (an exemplary sequence SEQ ED NO:l, SEQ ED NO:2, SEQ ED NO:3, SEQ ED NO:4, SEQ ED NO:5, SEQ ED NO:6, SEQ ED NO:7, SEQ ED NO:9, SEQ ED NO:10, SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, SEQ ED NO:14, SEQ ED NO:15, SEQ ED NO:16, SEQ ED NO:17, SEQ ED NO:18, SEQ ED NO:19, SEQ ID NO:20, SEQ TD NO:21, SEQ ED NO:22, SEQ ED NO:23, SEQ ED NO:24, SEQ ED NO:25, SEQ ED NO:26, SEQ TD NO:27, SEQ ED NO:28, SEQ ID NO:29, SEQ ED NO:30, SEQ ED NO:31, SEQ ED NO:12
- test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
- sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
- a "comparison window”, as used herein, includes reference to a segment of any one of the numbers of contiguous residues.
- continugous residues ranging anywhere from 20 to the full length of exemplary sequences SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:9, SEQ TD NO: 10, SEQ ED NO:ll, SEQ BD NO:12, SEQ BD NO:13, SEQ BD NO:14, SEQ BD NO:15, SEQ ED NO:16, SEQ ED NO:17, SEQ ED NO:18, SEQ ED NO:19, SEQ BD NO:20, SEQ ED NO:21, SEQ BD NO:22, SEQ ED NO:23, SEQ ED NO:24, SEQ ED NO:25, SEQ ED NO:26, SEQ BD NO:27, SEQ BD NO:28
- SEQ ED NO:3 SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:9, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95% sequence identity to SEQ ED NO:l, SEQ TD NO:3, SEQ TD NO:5, SEQ ED NO:7, SEQ BD NO:9, SEQ ED NO: 10, SEQ ED NO: 11, SEQ ED NO: 12, SEQ BD NO: 13, SEQ ED NO: 14, SEQ ED NO:15, SEQ TD NO:16, SEQ ID NO: 17, SEQ ED NO:18, SEQ ED NO: 19, SEQ ED NO:20, SEQ ED NO:21, SEQ ED NO:22, SEQ ED NO:23, SEQ ED NO:24, SEQ ED NO:25, SEQ TD NO:26, SEQ TD NO:27, SEQ ED NO:20, SEQ ED NO:21, SEQ
- subsequences ranging from about 20 to 600, about 50 to 200, and about 100 to 150 are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- Methods of alignment of sequence for comparison are well known in the art.
- Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of person & Lipman, Proc. Nat'l. Acad. Sci.
- Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences.
- a number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (Gibbs, 1995).
- Several genomes have been sequenced, e.g., M. genitalium (Fraser et al., 1995), M.jannaschii (Bult et al., 1996), H. influenzae (Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast (S. cerevisiae) (Mewes et al., 1997), and D.
- BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990) supra).
- HSPs high scoring sequence pairs
- initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
- the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
- the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc.
- BLAST Basic Local Alignment Search Tool
- five specific BLAST programs can be used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and, (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
- the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
- High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
- the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993).
- the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).
- the NCBI BLAST 2.2.2 programs is used, default options to blastp. There are about 38 setting options in the BLAST 2.2.2 program.
- all default values are used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a "-F F" setting is used, which disables filtering.
- Use of default filtering often results in Karlin- Altschul violations due to short length of sequence.
- Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
- the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention.
- the words "recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
- Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
- the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
- aspects of the invention include systems (e.g., internet based systems), particularly computer systems, which store and manipulate the sequences and sequence information described herein.
- a computer system 100 refers to the hardware components, software components, and data storage components used to analyze a nucleotide or polypeptide sequence of the invention.
- the computer system 100 can include a processor for processing, accessing and manipulating the sequence data.
- the processor 105 can be any well-known type of central processing unit, such as, for example, the Pentium ET from Intel Co ⁇ oration, or similar processor from Sun, Motorola, Compaq, AMD or International Business Machines.
- the computer system 100 is a general p pose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
- the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon.
- the computer system 100 can further include one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110.
- the data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or a modem capable of connection to a remote data storage system (e.g., via the internet) etc.
- the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon.
- the computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.
- the computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100.
- Software for accessing and processing the nucleotide or amino acid sequences of the invention can reside in main memory 115 during execution.
- the computer system 100 may further comprise a sequence comparison algorithm for comparing a nucleic acid sequence of the invention.
- the algorithm and sequence(s) can be stored on a computer readable medium.
- a "sequence comparison algorithm” refers to one or more programs which are implemented (locally or remotely) on the computer system 100 to compare a nucleotide sequence with other nucleotide sequences and/or compounds stored within a data storage means.
- the sequence comparison algorithm may compare the nucleotide sequences of the invention stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies or structural motifs. [00173]
- the parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied.
- Figure 9 is a flow diagram illustrating one aspect of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
- the database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK that is available through the Internet.
- the process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100.
- the memory could be any type of memory, including RAM or an internal storage device.
- the process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison.
- the process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer.
- a comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database.
- Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences.
- the parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system.
- a determination is made at a decision state 210 whether the two sequences are the same.
- the term "same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as "same" in the process 200. If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered.
- the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.
- one aspect of the invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid sequence of the invention and a sequence comparer for conducting the comparison.
- the sequence comparer may indicate a homology level between the sequences compared or identify stractural motifs, or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes.
- Figure 10 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.
- the process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory.
- the second sequence to be compared is then stored to a memory at a state 256.
- the process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read.
- the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U.
- sequence is a protein sequence, then it can be a single letter amino acid code so that the first and sequence sequences can be easily compared.
- a determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read.
- the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user.
- the level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with an every character in a second sequence, the homology level would be 100%.
- the computer program can compare a reference sequence to a sequence of the invention to determine whether the sequences differ at one or more positions.
- the program can record the length and identity of inserted, deleted or substituted nucleotides or amino acid residues with respect to the sequence of either the reference or the invention.
- the computer program may be a program which determines whether a reference sequence contains a single nucleotide polymo ⁇ hism (SNP) with respect to a sequence of the invention, or, whether a sequence of the invention comprises a SNP of a known sequence.
- the computer program is a program which identifies SNPs.
- the method may be implemented by the computer systems described above and the method illustrated in Figure 10.
- the computer based system comprises an identifier for identifying features within a nucleic acid or polypeptide of the invention.
- An "identifier" refers to one or more programs which identifies certain features within a nucleic acid sequence.
- an identifier may comprise a program which identifies an open reading frame (ORF) in a nucleic acid sequence.
- Figure 11 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence.
- the process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100.
- the process 300 then moves to a state 306 wherein a database of sequence features is opened.
- a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be "Initiation Codon" and the attribute would be "ATG”. Another example would be the feature name "TAATAA Box" and the feature attribute would be "TAATAA”.
- An example of such a database is produced by the University of Wisconsin Genetics Computer Group.
- the features may be stractural polypeptide motifs such as alpha helices, beta sheets, or functional polypeptide motifs such as enzymatic active sites, helix-tum-helix motifs or other motifs known to those skilled in the art.
- the process 300 moves to a state 308 wherein the first feature is read from the database.
- a comparison of the attribute of the first feature with the first sequence is then made at a state 310.
- a determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein • the name of the found feature is displayed to the user.
- the process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence. If the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.
- the invention provides a computer program that identifies open reading frames (ORFs).
- a polypeptide or nucleic acid sequence of the invention may be stored and manipulated in a variety of data processor programs in a variety of formats.
- a sequence can be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE.
- many computer programs and databases may be used as sequence comparison algorithms, identifiers, or sources of reference nucleotide sequences or polypeptide sequences to be compared to a nucleic acid sequence of the invention.
- the programs and databases used to practice the invention include, but are not limited to: MacPattem (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Bratlag et al. Comp. App. Biosci.
- Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
- the invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention, e.g., SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:9, SEQ ED NO:lO, SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, SEQ TD NO:14, SEQ ED NO:15, SEQ BD NO: 16, SEQ TD NO: 17, SEQ TD NO: 18, SEQ BD NO: 19, SEQ ED NO:20, SEQ ED NO:21, SEQ ED NO:22, SEQ DD NO:23, SEQ DD NO:24, SEQ ED NO:25, SEQ DD NO:26, SEQ DD NO:27, SEQ DD NO:28, SEQ ED NO:29, SEQ DD NO:30, SEQ DD NO:31, SEQ DD NO:31
- nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of a sequence of the invention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400 residues in length. Nucleic acids shorter than full length are also included. These nucleic acids are useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like.
- nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. [00180] Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency.
- a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45°C in a solution consisting of 0.9 M NaCl, 50 mM NaH 2 PO 4 , pH 7.0, 5.0 mM Na 2 EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 10 7 cpm (specific activity 4-9 X 10 8 cpm/ug) of 32 P end-labeled oligonucleotide probe are then added to the solution.
- the membrane is washed for 30 minutes at room temperature in IX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na 2 EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at T m -10°C for the oligonucleotide probe.
- IX SET 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na 2 EDTA
- the membrane is then exposed to auto-radiographic film for detection of hybridization signals.
- nucleic acids having different levels of homology to the probe can be identified and isolated.
- Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes.
- the melting temperature, T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe.
- Very stringent conditions are selected to be equal to or about 5°C lower than the T m for a particular probe.
- the melting temperature of the probe may be calculated using the following formulas:
- T m melting temperature
- Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 ⁇ g denatured fragmented salmon sperm DNA, 50% formamide.
- Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25°C below the T m .
- the hybridization may be conducted at 5-10°C below the T m .
- the hybridization is conducted at approximately 68°C.
- the hybridization is conducted at approximately 42°C.
- the filter is washed to remove any non-specifically bound detectable probe.
- the stringency used to wash the filters can also be varied depending on the nature of the nucleic acids being hybridized, the length of the nucleic acids being hybridized, the degree of complementarity, the nucleotide sequence composition (e.g., GC v. AT content), and the nucleic acid type (e.g., RNA v. DNA).
- Examples of progressively higher stringency condition washes are as follows: 2X SSC, 0.1% SDS at room temperature for 15 minutes (low stringency); 0.1X SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1X SSC, 0.5% SDS for 15 to 30 minutes at between the hybridization temperature and 68°C (high stringency); and 0.15M NaCl for 15 minutes at 72°C (very high stringency).
- a final low stringency wash can be conducted in 0.1X SSC at room temperature.
- the examples above are merely illustrative of one set of conditions that can be used to wash filters.
- One of skill in the art would know that there are numerous recipes for different stringency washes. Some other examples are given below.
- Nucleic acids which have hybridized to the probe are identified by autoradiography or other conventional techniques.
- the above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence.
- less stringent conditions may be used.
- the hybridization temperature maybe decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM.
- the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be “moderate” conditions above 50°C and "low” conditions below 50°C.
- a specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 55°C.
- a specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45°C.
- the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
- concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
- the filter may be washed with 6X SSC, 0.5% SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and "low” conditions below 25% formamide.
- 6X SSC 0.5% SDS at 50°C.
- the preceding methods may be used to isolate nucleic acids having a sequence with at least about 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50%) homology to a nucleic acid sequence of the invention, or fragments comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases thereof, and the sequences complementary thereto.
- Homology can be measured using an alignment algorithm.
- the homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic variants may have a substitution, deletion or addition of one or more nucleotides when compared to the nucleic acids of the invention or the sequences complementary thereto.
- wash conditions used to identify nucleic acids within the scope of the invention include, e.g.: a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50°C or about 55°C to about 60°C; or, a salt concentration of about 0.15 M NaCl at 72°C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 50°C or about 55°C to about 60°C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1X SSC containing 0.1% SDS at 68oC for 15 minutes; or, equivalent conditions.
- nucleic acid sequences of the invention are used as probes to identify and isolate related nucleic acids.
- the so-identified related nucleic acids may be cDNAs or genomic DNAs from organisms other than the one from which the nucleic acid of the invention was first isolated.
- a nucleic acid sample is contacted with the probe under conditions which permit the probe to specifically hybridize to related sequences. Hybridization of the probe to nucleic acids from the related organism is then detected using any of the methods described above.
- nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency.
- a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45°C in a solution consisting of 0.9 M NaCl, 50 mM NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 107 cpm (specific activity 4-9 X 108 cpm/ug) of 32P end-labeled oligonucleotide probe are then added to the solution.
- the membrane is washed for 30 minutes at room temperature (RT) in IX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at Tm-10°C for the oligonucleotide probe.
- IX SET 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA
- nucleic acids having different levels of homology to the probe can be identified and isolated.
- Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes.
- the melting temperature, Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe.
- Very stringent conditions are selected to be equal to or about 5°C lower than the Tm for a particular probe.
- the melting temperature of the probe may be calculated using the following exemplary formulas.
- Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm DNA, 50% formamide.
- Formulas for SSC and Denhardt's and other solutions are listed, e.g., in Sambrook.
- Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25°C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5-10°C below the Tm. In one aspect, hybridizations in 6X SSC are conducted at approximately 68°C. In one aspect, hybridizations in 50% formamide containing solutions are conducted at approximately 42°C. All of the foregoing hybridizations would be considered to be under conditions of high stringency.
- the filter is washed to remove any non- specifically bound detectable probe.
- the stringency used to wash the filters can also be varied depending on the nature of the nucleic acids being hybridized, the length of the nucleic acids being hybridized, the degree of complementarity, the nucleotide sequence composition (e.g., GC v. AT content), and the nucleic acid type (e.g., RNA v. DNA).
- Examples of progressively higher stringency condition washes are as follows: 2X SSC, 0.1% SDS at room temperature for 15 minutes (low stringency); 0.1X SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1X SSC, 0.5% SDS for 15 to 30 minutes at between the hybridization temperature and 68°C (high stringency); and 0.15M NaCl for 15 minutes at 72°C (very high stringency).
- a final low stringency wash can be conducted in 0. IX SSC at room temperature.
- the examples above are merely illustrative of one set of conditions that can be used to wash filters.
- One of skill in the art would know that there are numerous recipes for different stringency washes.
- Nucleic acids which have hybridized to the probe can be identified by autoradiography or other conventional techniques. The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate” conditions above 50°C and "low” conditions below 50°C.
- hybridization conditions are when the above hybridization is conducted at 55°C.
- low stringency hybridization conditions is when the above hybridization is conducted at 45°C.
- the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C. i this case, the concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
- the filter may be washed with 6X SSC, 0.5% SDS at 50°C.
- 6X SSC 0.5% SDS at 50°C.
- nucleic acids having a sequence with at least about 99%, 98%, 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% homology to a nucleic acid sequence of the invention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive bases thereof, and the sequences complementary thereto. Homology may be measured using an alignment algorithm, as discussed herein.
- the homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein.
- allelic variants may have a substitution, deletion or addition of one or more nucleotides when compared to a nucleic acid of the invention.
- the probes and methods of the invention may be used to isolate nucleic acids which encode polypeptides having at least about 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence identity (homology) to a polypeptide of the invention comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof as determined using a sequence alignment algorithm (e.g., such as the FASTA version 3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein). Oligonucleotides probes and methods for using them
- the invention also provides nucleic acid probes for identifying nucleic acids encoding a polypeptide with epoxide hydrolase activity.
- the probe comprises at least 10 consecutive bases of a sequence as set forth in an exemplary sequence of the invention.
- a probe of the invention can be at least about 5, 6, 7, 8 or 9 to about 40, about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence of the invention.
- the probes identify a nucleic acid by binding or hybridization.
- the probes can be used in arrays of the invention, see discussion below, including, e.g., capillary arrays.
- the probes of the invention can also be used to isolate other nucleic acids or polypeptides.
- the probes of the invention can be used to determine whether a biological sample, such as a soil sample, contains an organism having a nucleic acid sequence of the invention or an organism from which the nucleic acid was obtained.
- a biological sample potentially harboring the organism from which the nucleic acid was isolated is obtained and nucleic acids are obtained from the sample.
- the nucleic acids are contacted with the probe under conditions which permit the probe to specifically hybridize to any complementary sequences present in the sample.
- conditions which permit the probe to specifically hybridize to complementary sequences may be determined by placing the probe in contact with complementary sequences from samples known to contain the complementary sequence, as well as control sequences which do not contain the complementary sequence.
- Hybridization conditions such as the salt concentration of the hybridization buffer, the formamide concentration of the hybridization buffer, or the hybridization temperature, may be varied to identify conditions which allow the probe to hybridize specifically to complementary nucleic acids (see discussion on specific hybridization conditions).
- Hybridization may be detected by labeling the probe with a detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product.
- a detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product.
- more than one probe may be used in an amplification reaction to determine whether the sample contains an organism containing a nucleic acid sequence of the invention (e.g., an organism from which the nucleic acid was isolated).
- the probes comprise oligonucleotides.
- the amplification reaction may comprise a PCR reaction. PCR protocols are described in Ausubel and Sambrook (see discussion on amplification reactions). In such procedures, the nucleic acids in the sample are contacted with the probes, the amplification reaction is performed, and any resulting amplification product is detected.
- the amplification product may be detected by performing gel electrophoresis on the reaction products and staining the gel with an intercalator such as ethidium bromide.
- an intercalator such as ethidium bromide.
- one or more of the probes may be labeled with a radioactive isotope and the presence of a radioactive amplification product may be detected by autoradiography after gel electrophoresis.
- Probes derived from sequences near the 3' or 5' ends of a nucleic acid sequence of the invention can also be used in chromosome walking procedures to identify clones containing additional, e.g., genomic sequences. Such methods allow the isolation of genes which encode additional proteins of interest from the host organism.
- nucleic acid sequences of the invention are used as probes to identify and isolate related nucleic acids.
- the so-identified related nucleic acids may be cDNAs or genomic DNAs from organisms other than the one from which the nucleic acid of the invention was first isolated.
- a nucleic acid sample is contacted with the probe under conditions which permit the probe to specifically hybridize to related sequences. Hybridization of the probe to nucleic acids from the related organism is then detected using any of the methods described above.
- nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency.
- a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45°C in a solution consisting of 0.9 M NaCl, 50 mM NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 107 cpm (specific activity 4-9 X 108 cpm/ug) of 32P end-labeled oligonucleotide probe are then added to the solution.
- the membrane is washed for 30 minutes at room temperature (RT) in IX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at Tm-10°C for the oligonucleotide probe.
- IX SET 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA
- nucleic acids having different levels of homology to the probe can be identified and isolated.
- Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes.
- the melting temperature, Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe.
- Very stringent conditions are selected to be equal to or about 5°C lower than the Tm for a particular probe.
- the melting temperature of the probe may be calculated using the following exemplary formulas.
- Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOO ⁇ g denatured fragmented salmon sperm DNA, 50% formamide.
- Formulas for SSC and Denhardt's and other solutions are listed, e.g., in Sambrook.
- Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25°C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5-10°C below the Tm. In one aspect, hybridizations in 6X SSC are conducted at approximately 68°C. In one aspect, hybridizations in 50% formamide containing solutions are conducted at approximately 42°C. All of the foregoing hybridizations would be considered to be under conditions of high stringency.
- the filter is washed to remove any non-specifically bound detectable probe.
- the stringency used to wash the filters can also be varied depending on the nature of the nucleic acids being hybridized, the length of the nucleic acids being hybridized, the degree of complementarity, the nucleotide sequence composition (e.g., GC v. AT content), and the nucleic acid type (e.g., RNA v. DNA).
- Examples of progressively higher stringency condition washes are as follows: 2X SSC, 0.1% SDS at room temperature for 15 minutes (low stringency); 0.1X SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1X SSC, 0.5% SDS for 15 to 30 minutes at between the hybridization temperature and 68°C (high stringency); and 0.15M NaCl for 15 minutes at 72°C (very high stringency).
- a final low stringency wash can be conducted in 0.1X SSC at room temperature.
- the examples above are merely illustrative of one set of conditions that can be used to wash filters.
- One of skill in the art would know that there are numerous recipes for different stringency washes.
- Nucleic acids which have hybridized to the probe can be identified by autoradiography or other conventional techniques. The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate” conditions above 50°C and "low” conditions below 50°C.
- hybridization conditions are when the above hybridization is conducted at 55°C.
- low stringency hybridization conditions is when the above hybridization is conducted at 45°C.
- the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
- the concentration of formamide in the hybridization buffer maybe reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
- the filter may be washed with 6X SSC, 0.5% SDS at 50°C.
- 6X SSC 0.5% SDS at 50°C.
- nucleic acids having a sequence with at least about 99%, 98%, 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, .at least 60%, at least 55%, or at least 50%) homology to a nucleic acid sequence of the invention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive bases thereof, and the seq ⁇ ences complementary thereto.
- homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein.
- allelic variants may have a substitution, deletion or addition of one or more nucleotides when compared to a nucleic acid of the invention.
- the probes and methods of the invention may be used to isolate nucleic acids which encode polypeptides having at least about 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence identity (homology) to a polypeptide of the invention comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof as determined using a sequence alignment algorithm (e.g., such as the FASTA version 3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein).
- sequence alignment algorithm e.g., such as the FASTA version 3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein.
- the invention further provides for nucleic acids complementary to (e.g., antisense sequences to) the nucleic acid sequences of the invention.
- Antisense sequences are capable of inhibiting the transport, splicing or transcription of epoxide hydrolase- encoding genes. The inhibition can be effected through the targeting of genomic DNA or messenger RNA. The transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage.
- One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind epoxide hydrolase gene or message, in either case preventing or inhibiting the production or function of epoxide hydrolase. The association can be through sequence specific hybridization.
- Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of epoxide hydrolase message.
- the oligonucleotide can have enzyme activity wliich causes such cleavage, such as ribozymes.
- the oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One may screen a pool of many different such oligonucleotides for those with the desired activity.
- the invention provides antisense oligonucleotides capable of binding epoxide hydrolase message which can inhibit proteolytic activity by targeting mRNA.
- Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such epoxide hydrolase oligonucleotides using the novel reagents of the invention.
- gene walking/ RNA mapping protocols to screen for effective antisense oligonucleotides are well known in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183, describing an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection. See also Smith (2000) Eur. J. Pharm. Sci. 11:191-198.
- Naturally occurring nucleic acids are used as antisense oligonucleotides.
- the antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening.
- the antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening.
- a wide variety of synthetic, non- naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem.
- peptide nucleic acids containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used.
- Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189D197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).
- Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and mo ⁇ holino carbamate nucleic acids, as described above.
- Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense epoxide hydrolase sequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
- Inhibitory Ribozymes [00222] The invention provides for with ribozymes capable of binding epoxide hydrolase message which can inhibit proteolytic activity by targeting mRNA.
- Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme wliich is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA.
- the ribozyme recognizes and binds a target RNA through complementary basepairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA.
- the enzymatic nature of a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide.
- a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
- the enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hai ⁇ in, hepatitis delta virus, group I intron or RnaseP-like RNA (in association with an RNA guide sequence).
- hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hai ⁇ in motifs byHampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.
- RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule.
- the invention provides methods of generating variants of the nucleic acids of the invention, e.g., those encoding an epoxide hydrolase enzyme. These methods can be repeated or used in various combinations to generate epoxide hydrolase enzymes having an altered or different activity or an altered or different stability from that of an epoxide hydrolase encoded by the template nucleic acid. These methods also can be repeated or used in various combinations, e.g., to generate variations in gene/ message expression, message translation or message stability.
- the genetic composition of a cell is altered by, e.g., modification of a homologous gene ex vivo, followed by its reinsertion into the cell.
- a nucleic acid of the invention can be altered by any means. For example, random or stochastic methods, or, non-stochastic, or "directed evolution,” methods, see, e.g., U.S. Patent No. 6,361,974. Methods for random mutation of genes are well known in the art, see, e.g., U.S. Patent No. 5,830,696. For example, mutagens can be used to randomly mutate a gene.
- Mutagens include, e.g., ultraviolet light or gamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to induce DNA breaks amenable to repair by recombination.
- Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
- Other mutagens are analogues of nucleotide precursors, e.g., nitrosoguanidine, 5-bromouracil, 2-ammopurine, or acridine. These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.
- any technique in molecular biology can be used, e.g., random PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471; or, combinatorial multiple cassette mutagenesis, see, e.g., Crameri (1995) Biotechniques 18:194-196.
- nucleic acids e.g., genes
- modifications, additions or deletions are introduced by error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil- containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic
- Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling et al. (1997) “Approaches to DNA mutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al. (1996) “Oligonucleotide-directed random mutagenesis using the phosphorothioate method” Methods Mol. Biol. 57:369-374; Smith
- Additional protocols used in the methods of the invention include point mismatch repair (Kramer (1984) "Point Mismatch Repair” Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al. (1985) "Improved oligonucleotide site- directed mutagenesis using M13 vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using Ml 3 vectors” Methods in Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) "Use of oligonucleotides to generate large deletions" Nucl.
- Non-stochastic, or "directed evolution,” methods include, e.g., saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof are used to modify the nucleic acids of the invention to generate epoxide hydrolase with new or altered properties (e.g., activity under highly acidic or alkaline conditions, high temperatures, and the like).
- Polypeptides encoded by the modified nucleic acids can be screened for an activity before testing for proteolytic or other activity. Any testing modality or protocol can be used, e.g., using a capillary array platform. See, e.g., U.S. Patent Nos. 6,361,974; 6,280,926; 5,939,250. Saturation mutagenesis, or, GSSM
- non-stochastic gene modification a "directed evolution process” is used to generate epoxide hydrolases with new or altered properties. Variations of this method have been termed “gene site-saturation mutagenesis,” “site- saturation mutagenesis,” “saturation mutagenesis” or simply “GSSM.” It can be used in combination with other mutagenization processes. See, e.g., U.S. Patent Nos. 6,171,820; 6,238,884.
- GSSM comprises providing a template polynucleotide and a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide with the oligonucleotides, thereby generating polynucleotides comprising homologous gene sequence variations.
- codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amino acid residue in an enzyme active site or ligand binding site targeted to be modified.
- These oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N,N,G/T sequence, and, optionally, a second homologous sequence.
- the downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids.
- one such degenerate oligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions.
- At least two degenerate cassettes are used - either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.
- more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site.
- This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).
- oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.
- simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence.
- degenerate cassettes having less degeneracy than the N,N,G/T sequence are used. For example, it may be desirable in some instances to use (e.g.
- a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet.
- Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet.
- degenerate triplets allows for systematic and easy generation of a full range of possible natural amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide (in alternative aspects, the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated.
- an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet 32 individual sequences can code for all 20 possible natural amino acids.
- a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using at least one such oligonucleotide there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides.
- the use of a non-degenerate oligonucleotide in site- directed mutagenesis leads to only one progeny polypeptide product per reaction vessel.
- Nondegenerate oligonucleotides can optionally be used in combination with degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides one means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.
- each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide (e.g., epoxide hydrolases) molecules such that all 20 natural amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations).
- the 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening.
- an individual progeny polypeptide When an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased proteolytic activity under alkaline or acidic conditions), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein. [00238] In one aspect, upon mutagenizing each and every amino acid position in a parental polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes may be identified at more than one amino acid position. One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions.
- site-saturation mutagenesis can be used together with another stochastic or non-stochastic means to vary sequence, e.g., synthetic ligation reassembly (see below), shuffling, chimerization, recombination and other mutagenizing processes and mutagenizing agents.
- synthetic ligation reassembly see below
- shuffling chimerization
- recombination recombination and other mutagenizing processes and mutagenizing agents.
- This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.
- the invention provides a non-stochastic gene modification system termed "synthetic ligation reassembly,” or simply “SLR,” a “directed evolution process,” to generate epoxide hydrolases with new or altered properties.
- SLR is a method of ligating oligonucleotide fragments together non-stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non- stochastically. See, e.g., U.S. Patent Application Serial No.
- SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucle
- SLR does not depend on the presence of high levels of homology between polynucleotides to be rearranged.
- this method can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10100 different chimeras.
- SLR can be used to generate libraries comprised of over 101000 different progeny chimeras.
- aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecule shaving an overall assembly order that is chosen by design. This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.
- the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.
- the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends. If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).
- the annealed building pieces are treated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent bonding of the building pieces.
- a ligase e.g. T4 DNA ligase
- the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates that serve as a basis for producing a progeny set of finalized chimeric polynucleotides.
- These parental oligonucleotide templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, e.g., chimerized or shuffled.
- the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points.
- the demarcation points can be located at an area of homology, and are comprised of one or more nucleotides. These demarcation points are preferably shared by at least two of the progenitor templates. The demarcation points can thereby be used to delineate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides.
- the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules.
- a demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences.
- a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences. Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences. In one aspect, a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.
- a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides.
- all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.
- the assembly order i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
- the assembly order is by design (or non-stochastic) as described above. Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.
- the ligation reassembly method is performed systematically.
- the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g. one by one.
- this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups.
- the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.
- the saturation mutagenesis and optimized directed evolution methods also can be used to generate different progeny molecular species.
- the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template. Alternatively, a codon can be altered such that the coding for an originally amino acid is altered.
- This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecular homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.
- the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g. by mutagenesis) or in an in vivo process (e.g. by utilizing the gene splicing ability of a host organism).
- nucleotides e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences
- a nucleic acid building block is used to introduce an intron.
- functional introns are introduced into a man-made gene manufactured according to the methods described herein.
- the artificially introduced intron(s) can be functional in a host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.
- the invention provides a non-stochastic gene modification system termed "optimized directed evolution system" to generate epoxide hydrolases with new or altered properties.
- Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination.
- Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
- a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.
- This method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
- this method provides a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.
- One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides corresponding to fragments or portions of each parental sequence. Each oligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order.
- oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.
- three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.
- a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences.
- each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
- a probability density function can be determined to predict the population of crossover events that are likely to occur during «ach step in a ligation reaction given a set number of parental variants, a number of oligonucleotides corresponding to each variant, and the concentrations of each variant during each step in the ligation reaction.
- PDF probability density function
- a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events.
- These methods are directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of a nucleic acid encoding a polypeptide through recombination.
- This system allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
- a crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.
- the method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
- these methods provide a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.
- the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events.
- each of the molecules chosen for further analysis most likely has, for example, only three crossover events.
- the boundaries on the functional variety between the chimeric molecules is reduced. This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.
- the method creates a chimeric progeny polynucleotide sequence by creating oligonucleotides corresponding to fragments or portions of each parental sequence.
- Each oligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. See also USSN 09/332,835.
- the number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.
- three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.
- a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences. The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low.
- each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
- a probability density function can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides corresponding to each variant, and the concentrations of each variant during each step in the ligation reaction.
- PDF probability density function
- a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events. Determining Crossover Events
- aspects of the invention include a system and software that receive a desired crossover probability density function (PDF), the number of parent genes to be reassembled, and the number of fragments in the reassembly as inputs.
- PDF crossover probability density function
- the output of this program is a "fragment PDF" that can be used to determine a recipe for producing reassembled genes, and the estimated crossover PDF of those genes.
- the processing described herein is preferably performed in MATLABa (The Mathworks, Natick, Massachusetts) a programming language and development environment for technical computing. Iterative Processes
- these processes can be iteratively repeated.
- a nucleic acid (or, the nucleic acid) responsible for an altered epoxide hydrolase phenotype is identified, re-isolated, again modified, re-tested for activity.
- This process can be iteratively repeated until a desired phenotype is engineered.
- an entire biochemical anabolic or catabolic pathway can be engineered into a cell, including proteolytic activity.
- oligonucleotide has no affect at all on the desired trait (e.g., a new epoxide hydrolase phenotype)
- it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed. Since inco ⁇ orating the sequence within a larger sequence prevents any crossover events, there will no longer be any variation of this sequence in the progeny polynucleotides.
- This iterative practice of determining which oligonucleotides are most related to the desired trait, and which are unrelated allows more efficient exploration all of the possible protein variants that might be provide a particular trait or activity. In vivo shuffling
- In vivo shuffling of molecules is use in methods of the invention that provide variants of polypeptides of the invention, e.g., antibodies, epoxide hydrolases, and the like.
- In vivo shuffling can be performed utilizing the natural property of cells to recombine multimers. While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.
- the invention provides a method for producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide.
- the invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell.
- the regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide.
- hybrid polynucleotide is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences. Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules. In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule. Producing sequence variants
- the invention also provides methods of making sequence variants of the nucleic acid and epoxide hydrolase sequences of the invention or isolating epoxide hydrolases using the nucleic acids and polypeptides of the invention.
- the invention provides for variants of an epoxide hydrolase gene of the invention, which can be altered by any means, including, e.g., random or stochastic methods, or, non- stochastic, or "directed evolution," methods, as described above.
- the isolated variants may be naturally occurring. Variant can also be created in vitro.
- Variants may be created using genetic engineering techniques such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives may be created using chemical synthesis or modification procedures. Other methods of making variants are also familiar to those skilled in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids which encode polypeptides having characteristics which enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. These nucleotide differences can result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
- variants may be created using error prone PCR.
- error prone PCR PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product.
- Error prone PCR is described, e.g., in Leung, D.W., et al., Technique, 1:11-15, 1989) and Caldwell, R. C. & Joyce G.F., PCR Methods Applic, 2:28-33, 1992.
- nucleic acids to be mutagenized are mixed with PCR primers, reaction buffer, MgC12, MnC12, Taq polymerase and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product.
- the reaction may be performed using 20 frnoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50mM KCl, lOmM Tris HC1 (pH 8.3) and 0.01% gelatin, 7mM MgC12, 0.5mM MnC12, 5 units of Taq polymerase, 0.2mM dGTP, 0.2mM dATP, ImM dCTP, and ImM dTTP. PCR may be performed for 30 cycles of 94° C for 1 min, 45° C for 1 min, and 72° C for 1 min. However, it will be appreciated that these parameters may be varied as appropriate.
- the mutagenized nucleic acids are cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids is evaluated.
- Variants may also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized. Clones containing the mutagenized DNA are recovered and the activities of the polypeptides they encode are assessed. [00266] Another method for generating variants is assembly PCR.
- Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, e.g., U.S. Patent No. 5,965,408.
- Still another method of generating variants is sexual PCR mutagenesis.
- sexual PCR mutagenesis forced homologous recombination occurs between DNA molecules of different but highly related DNA sequence in vitro, as a result of random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction.
- Sexual PCR mutagenesis is described, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, in such procedures a plurality of nucleic acids to be recombined are digested with DNase to generate fragments having an average size of 50-200 nucleotides.
- Fragments of the desired average size are purified and resuspended in a PCR mixture.
- PCR is conducted under conditions which facilitate recombination between the nucleic acid fragments.
- PCR may be performed by resuspending the purified fragments at a concentration of 10-30ng/:l in a solution of 0.2mM of each dNTP, 2.2mM MgC12, 50mM KCL, lOmM Tris HC1, pH 9.0, and 0.1% Triton X-100.
- PCR 2.5 units of Taq polymerase per 100:1 of reaction mixture is added and PCR is performed using the following regime: 94° C for 60 seconds, 94°C for 30 seconds, 50-55° C for 30 seconds, 72° C for 30 seconds (30-45 times) and 72°C for 5 minutes.
- oligonucleotides may be included in the PCR reactions.
- the Klenow fragment of DNA polymerase I may be used in a first set of PCR reactions and Taq polymerase may be used in a subsequent set of PCR reactions. Recombinant sequences are isolated and the activities of the polypeptides they encode are assessed.
- Variants may also be created by in vivo mutagenesis.
- random mutations in a sequence of interest are generated by propagating the sequence of interest in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways.
- a bacterial strain such as an E. coli strain
- Such "mutator" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA.
- Mutator strains suitable for use for in vivo mutagenesis are described, e.g., in PCT Publication No. WO 91/16427.
- Variants may also be generated using cassette mutagenesis.
- cassette mutagenesis a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide often contains completely and/or partially randomized native sequence.
- Recursive ensemble mutagenesis may also be used to generate variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis.
- Recursive ensemble mutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815. [00271] In some aspects, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described, e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random and site-directed mutagenesis are described, e.g., in Arnold (1993) Current Opinion in Biotechnology 4:450-455.
- the variants are created using shuffling procedures wherein portions of a plurality of nucleic acids which encode distinct polypeptides are fused together to create chimeric nucleic acid sequences which encode chimeric polypeptides as described in, e.g., U.S. Patent Nos. 5,965,408; 5,939,250.
- the invention also provides variants of polypeptides of the invention comprising sequences in which one or more of the amino acid residues (e.g., of an exemplary polypeptide, such as SEQ ID NO:2) are substituted with a conserved or non- conserved amino acid residue (e.g., a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
- Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics.
- polypeptides of the invention include those with conservative substitutions of sequences of the invention, e.g., the exemplary SEQ ED NO:2, including but not limited to the following replacements: replacements of an aliphatic amino acid such as Alanine, Valine, Leucine and Isoleucine with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue such as Aspartic acid and Glutamic acid with another acidic residue; replacement of a residue bearing an amide group, such as Asparagine and Glutamine, with another residue bearing an amide group; exchange of a basic residue such as Lysine and Arginine with another basic residue; and replacement of an aromatic residue such as Phenylalanine, Tyrosine with another aromatic residue.
- Other variants are those in which one or more of the amino acid residues of the polypeptides of the invention includes a substituent group.
- variants within the scope of the invention are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide, for example, polyethylene glycol.
- additional variants within the scope of the invention are those in which additional amino acids are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.
- the variants, fragments, derivatives and analogs of the polypeptides of the invention retain the same biological function or activity as the exemplary polypeptides, e.g., a proteolytic activity, as described herein.
- the variant, fragment, derivative, or analog includes a proprotein, such that the variant, fragment, derivative, or analog can be activated by cleavage of the proprotein portion to produce an active polypeptide.
- the invention provides methods for modifying epoxide hydrolase-encoding nucleic acids to modify codon usage.
- the invention provides methods for modifying codons in a nucleic acid encoding an epoxide hydrolase to increase or decrease its expression in a host cell.
- the invention also provides nucleic acids encoding an epoxide hydrolase modified to increase its expression in a host cell, epoxide hydrolase so modified, and methods of making the modified epoxide hydrolases.
- the method comprises identifying a "non-preferred” or a "less preferred” codon in epoxide hydrolase- encoding nucleic acid and replacing one or more of these non-preferred or less preferred codons with a "preferred codon” encoding the same amino acid as the replaced codon and at least one non-preferred or less preferred codon in the nucleic acid has been replaced by a preferred codon encoding the same amino acid.
- a preferred codon is a codon over- represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell.
- Host cells for expressing the nucleic acids, expression cassettes and vectors of the invention include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Thus, the invention provides methods for optimizing codon usage in all of these cells, codon-altered nucleic acids and polypeptides made by the codon-altered nucleic acids.
- Exemplary host cells include gram negative bacteria, such as Escherichia coli and Pseudomonas fluorescens; gram positive bacteria, such as Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis.
- Exemplary host cells also include eukaryotic organisms, e.g., various yeast, such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis, Hansenula polymo ⁇ ha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines.
- yeast such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis, Hansenula polymo ⁇ ha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines.
- yeast such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces
- the codons of a nucleic acid encoding an epoxide hydrolase isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell different from the bacteria from which the epoxide hydrolase was derived, a yeast, a fungi, a plant cell, an insect cell or a mammalian cell.
- Methods for optimizing codons are well known in the art, see, e.g., U.S. Patent No. 5,795,737; Baca (2000) Int. J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188; Naram (2001) Infect. Immun.
- the invention provides transgenic non-human animals comprising a nucleic acid, a polypeptide (e.g., epoxide hydrolase), an expression cassette or vector or a transfected or transformed cell of the invention.
- the transgenic non-human animals can be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice, comprising the nucleic acids of the invention. These animals can be used, e.g., as in vivo models to study epoxide hydrolase activity, or, as models to screen for modulators of epoxide hydrolase activity in vivo.
- the coding sequences for the polypeptides to be expressed in the transgenic non- human animals can be designed to be constitutive, or, under the control of tissue-specific, developmental-specific or inducible transcriptional regulatory factors.
- Transgenic non- human animals can be designed and generated using any method known in the art; see, e.g., U.S. Patent Nos.
- U.S. Patent No. 6,211,4208 describes making and using transgenic non-human mammals which express in their brains a nucleic acid construct comprising a DNA sequence.
- U.S. Patent No. 5,387,742 describes injecting cloned recombinant or synthetic DNA sequences into fertilized mouse eggs, implanting the injected eggs in pseudo-pregnant females, and growing to term transgenic mice whose cells express proteins related to the pathology of Alzheimer's disease.
- U.S. Patent No. 6,187,992 describes making and using a transgenic mouse whose genome comprises a disraption of the gene encoding amyloid precursor protein (APP).
- APP amyloid precursor protein
- the transgenic or modified animals of the invention can also be used to practice the methods of the invention.
- the transgenic or modified animals of the invention comprise a "knockout animal,” e.g., a “knockout mouse,” engineered not to express or to be unable to express an epoxide hydrolase.
- the invention provides isolated or recombinant polypeptides having a sequence identity to an exemplary sequence of the invention, e.g., SEQ ED NO:2; SEQ ED NO:4; SEQ ED NO:6; SEQ ED NO:8, SEQ ED NO: 10, SEQ ED NO:12, SEQ ED NO:14, SEQ ID NO:16, SEQ TD NO:18, SEQ ED NO:20; SEQ ED NO:22; SEQ ED NO:22; SEQ ED NO:26; SEQ ED NO:28, SEQ BD NO:30, SEQ TD NO:32, SEQ ID NO:34, SEQ ID NO:36; SEQ ID NO:38, SEQ HD NO:40, SEQ ED NO:42, SEQ ED NO:44, SEQ ED NO:46, SEQ ED NO:48, SEQ TD NO:50; SEQ ED NO:52; SEQ ID NO:54; SEQ HD NO:56
- the identity can be over the full length of the polypeptide, or, the identity can be over a region of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues.
- Polypeptides of the invention can also be shorter than the full length of exemplary polypeptides (e.g., SEQ ID NO:2; SEQ HD NO:4; SEQ TD NO:6; SEQ ID NO:8, SEQ VD NO:10, SEQ ID NO:12, SEQ ED NO:14, SEQ DD NO:16; SEQ DD NO:18, SEQ DD NO:20; SEQ ID NO:22; SEQ ID NO:22; SEQ VD NO:26; SEQ HD NO:28, SEQ HD NO:30, SEQ ID NO:32, SEQ DD NO:34, SEQ DD NO:36; SEQ DD NO:38, SEQ DD NO:40, SEQ DD NO:42, SEQ TD NO:44, SEQ ED NO:46, SEQ DD NO:48, SEQ DD NO:50; SEQ DD NO:52; SEQ DD NO:54; SEQ DD NO:56; SEQ DD NO:58, SEQ TD
- the invention provides polypeptides (peptides, fragments) ranging in size between about 5 and the full length of a polypeptide, e.g., an enzyme, such as an epoxide hydrolase; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of an exemplary epoxide hydrolase of the invention.
- an enzyme such as an epoxide hydrolase
- exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous
- Peptides of the invention can be useful as, e.g., labeling probes, antigens, toleragens, motifs, epoxide hydrolase active sites.
- Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo.
- the peptides and polypeptides of the invention can be made and isolated using any method known in the art.
- Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Carathers (1980) Nucleic Acids Res. Symp.
- peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis maybe achieved, e.g., using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
- the peptides and polypeptides of the invention can also be glycosylated.
- the glycosylation can be added post-translationally either chemically or by cellular biosynthetic mechanisms, wherein the later inco ⁇ orates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence.
- the glycosylation can be O-linked or N-linked.
- the peptides and polypeptides of the invention, as defined above, include all "mimetic" and "peptidomimetic" forms.
- mimetic and “peptidomimetic” refer to a synthetic chemical compound which has substantially the same stractural and/or functional characteristics of the polypeptides of the invention.
- the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
- the mimetic can also inco ⁇ orate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
- routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and or function is not substantially altered.
- a mimetic composition is within the scope of the invention if it has an epoxide hydrolase activity.
- Polypeptide mimetic compositions of the invention can contain any combination of non-natural structural components.
- mimetic compositions of the invention include one or all of the following three stractural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
- a secondary structural mimicry i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
- a polypeptide of the invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
- Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'- diisopropylcarbodiimide (DIC).
- a polypeptide of the invention can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues.
- Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
- Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L- phenylglycine; D- or L- 2 thieneylalanine; D- or L-l, -2, 3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3- ⁇ yridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D- (trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p- biphenylphenylalanine; K- or L-p-methoxy-biphenylphen
- Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
- Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
- Carboxyl side groups e.g., aspartyl or glutamyl
- Carboxyl side groups can also be selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as, e.g., 1- cyclohexyl-3(2-mo ⁇ holinyl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia- 4,4- dimetholpentyl) carbodiimide.
- Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
- Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids omithine, citralline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.
- Nitrile derivative e.g., containing the CN-moiety in place of COOH
- Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
- Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclo- hexanedione, or ninhydrin, preferably under alkaline conditions.
- Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O- acetyl tyrosyl species and 3-nitro derivatives, respectively.
- Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
- alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and corresponding amines
- Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole.
- cysteinyl residues e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid
- chloroacetyl phosphate N-alkylmaleimides
- 3-nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
- Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro- benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide.
- Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4- hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,- dimethylproline.
- Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
- mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
- a residue, e.g., an amino acid, of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality.
- any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical stractural type or a peptidomimetic, but of the opposite chirality, referred to as the D- amino acid, but also can be referred to as the R- or S- form.
- the invention also provides methods for modifying the polypeptides of the invention by either natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc), or by chemical modification techniques, and the resulting modified polypeptides. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications.
- Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation.
- Solid-phase chemical peptide synthesis methods can also be used to synthesize the polypeptide or fragments of the invention. Such method have been known in the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc, 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J.
- a plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the pin's or rod's tips.
- amino acids are built into desired peptides.
- FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an Applied Biosystems, Inc. Model 431 ATM automated peptide synthesizer. Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.
- Epoxide hydrolases show promise as appealing tools for the synthesis of enantiopure epoxides via the hydrolytic kinetic resolution of racemic epoxides. Some of the attractive features of this potentially useful enzyme class are outlined below.
- EHs are ubiquitous in nature. EHs have been found in all mammalian species tested, with the mammalian liver microsomal epoxide hydrolase (mEH) being the best studied. (Armstrong, R. N. Drug Metab. Rev. 1999, 31, 71-86.) Most mammalian EHs are involved in the detoxification of epoxides, while a few are engaged in the biosynthesis of hormones.
- EHs have been found from various bacteria, yeast, and fungi.
- Examples of bacterial EHs include those isolated from Agrobacterium radiobacter, Rhodococcus sp., Corynebacterium sp., Mycobacterium paraffinicum, Nocardia sp., Pseudomonas NRRL B-2994, and some Streptomyces strains.
- Fungal EHs were also found in Aspergillus niger, Helminthosporum sativum, Diploida gossypina, Beauveria sulfurescens, and some Fursarium strains.
- the best-known yeast EH is Rhodotorula glutinis enzyme. Almost all of these enzymes were discovered during the screening of available strains with various epoxide substrates, and only a handful of them were further investigated at the genetic level. Some of these enzymes showing good enantioselectivity and potentially being readily available through fermentation.
- the scope of substrates recognized by microbial epoxide hydrolases need to be expanded and discovery of novel EHs should offer a viable solution.
- EHs are cofactor-free, 'easy-to-use' catalysts. Biochemical studies have shown that EHs, like other well-recognized hydrolytic enzymes such as lipases and esterases, require neither prosthetic groups nor metal ions for activity. Current proposed mechanism by wliich EHs operate also bears similarity to that of esterases in that a covalent adduct is formed between the enzyme active site and the substrate during the catalytic cycle. Site-directed mutagenesis studies and structural data of a bacterial enzyme (A. radiobacter) suggested an active site Asp as the nucleophile. (Nardini, M.; Ridder, I. S.; Rozeboon, H. J.; Kalk, K. H.; Rink, R.; Janssen, D. B.; Dijkstra, B. W. J. Biol. Chem. 1999, 274, 14579-14596.)
- FIG. 12 illustrates the mechanism of A. radiobacter epoxide hydrolase.
- the catalytic mechanism involves two distinct steps.
- the first step (a) is an SN2 nucleophihc attack by an Asp 107 carboxylate oxygen on the least hindered carbon atom of the epoxide, resulting in a covalent ester intermediate.
- the ester intermediate is hydrolyzed by a water molecule that is activated by the Asp246-His275 pair.
- epoxide hydrolysis has important stereochemical consequences: the regioselectivity (two possible carbons being attacked) and the inversion of absolute configuration at the attacked carbon. Therefore, both the regioselectivity and enantioselectivity need to be taken into consideration when analyzing epoxide hydrolase-catalyzed reactions.
- EHs often exhibit high enantioselectivity as well as high activity toward certain categories of epoxide substrates.
- epoxide substrates can be divided into five types: mono-substituted, 2,2-disubstituted, 2,3-disubstituted, trisubstituted, and styrene-oxides ( Figure 13).
- the known EHs have been shown to have different stereoselectivities to different types of substrates.
- Norcardia EH1 catalyzed the enantioconvergent hydrolysis of cis-2,3-epoxyheptane to 2R,3R-2,3-dihydroxyheptane with good yield and enantiomeric excess (Figure 14).
- Styrene-oxides are viewed as a special group of substrates because the benzylic carbon of these substrates provides stability to the carbocation nature of the transition state of the reaction. As a result, this group of substrates usually exhibits poor regioselectivity if the benzylic carbon is also sterically hindered. However, excellent enantioselectivity was observed in the reactions catalyzed by enzymes from red yeasts such as Rhodotorala glutinis strain CTMW 147, and in particular the fungal epoxide hydrolases, such as the enzyme from Aspergillus niger. (Weijers, C. A. G. M.
- Chiral epoxides and diols have important applications in anti-cancer, antivirals, antifungals, antibacterials, and other pharmaceuticals.
- epoxide hydrolases have shown great promise. As a kinetic resolution method with a limit of 50% yield, epoxide hydrolase-mediated syntheses are not expected to completely replace the current chemical asymmetric epoxidation.
- epoxide hydrolases can be envisioned in the following capacities: to replace chemical methods as "cleaner" catalysts in certain transformations; to be the choice of catalysts where the chemical methods are limited; to prepare certain diols in an enantioconvergent manner where the yields are not limited to 50%; to be used in combination with other asymmetric epoxidation methods to improve overall ee value by hydrolyzing a minor epoxide enantiomer.
- the bioactivity of interest is activity as a catalyst for the modification of epoxides.
- biomolecule refers to epoxide hydrolases.
- the first step of the efforts for discovering these enzymes involves developing sensitive, high throughput methods for the discovery of catalysts for the modification of epoxides.
- a combination of optimized assays and screening hosts can be applied to demonstrate that biocatalysts can be obtained from environmental gene libraries.
- the host strain libraries and environmental gene libraries can be built using the technologies described in U.S. Patent No. 5,958,672, U.S. Patent No. 6,001,574 and U.S. Patent No. 5,763,239.
- the invention provides hybrid epoxide hydrolases and fusion proteins, including peptide libraries, comprising sequences of the invention.
- the peptide libraries comprising sequences of the invention are used to isolate peptide inhibitors of targets (e.g., receptors, enzymes) and to identify formal binding partners of targets (e.g., ligands, such as cytokines, hormones and the like).
- biomolecule screening for biologically and therapeutically relevant compounds is rapidly growing.
- Relevant biomolecules that have been the focus of such screening include chemical libraries, nucleic acid libraries and peptide libraries, in search of molecules that either inhibit or augment the biological activity of identified target molecules.
- peptide libraries the isolation of peptide inhibitors of targets and the identification of formal binding partners of targets has been a key focus. Screening of combinatorial libraries of potential drags on therapeutically relevant target cells is a rapidly growing and important field.
- one particular problem with peptide libraries is the difficulty assessing whether any particular peptide has been expressed, and at what level, prior to determining whether the peptide has a biological effect.
- the peptides need to be expressed in sufficient quantities to overcome catabolic mechanisms such as proteolysis and transport out of the cytoplasm into endosomes.
- the fusion proteins of the invention e.g., the peptide moiety
- the fusion proteins of the invention are conformationally stabilized (relative to linear peptides) to allow a higher binding affinity for their cellular targets.
- the present invention provides fusions of epoxide hydrolases of the invention and other peptides, including known and random peptides, that are fused in such a manner that the structure of the epoxide hydrolases is not significantly perturbed and the peptide is metabolically or structurally conformationally stabilized. This allows the creation of a peptide library that is easily monitored, both for its presence within cells and its quantity.
- Amino acid sequence variants of the invention can be characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the epoxide hydrolase amino acid sequence.
- the variants of the invention exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics. While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed epoxide hydrolase variants screened for the optimal combination of desired activity.
- amino acid substitutions can be single residues; insertions can be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated.
- Deletions can range from about 1 to about 20 residues, although in some cases deletions may be much larger.
- substitutions, deletions, insertions or any combination thereof may be used. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.
- the invention provides epoxide hydrolases where the structure of the polypeptide backbone, the secondary or the tertiary structure, e.g., an alpha-helical or beta-sheet structure, has been modified.
- the charge or hydrophobicity has been modified.
- the bulk of a side chain has been modified.
- Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
- substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g.
- variants can exhibit the same qualitative biological activity (i.e. proteolytic activity) although variants can be selected to modify the characteristics of the epoxide hydrolases as needed.
- epoxide hydrolases of the invention comprise epitopes or purification tags, signal sequences or other fusion sequences, etc.
- the epoxide hydrolases of the invention can be fused to a random peptide to form a fusion polypeptide.
- fused or “operably linked” herein is meant that the random peptide and the epoxide hydrolase are linked together, in such a manner as to minimize the disruption to the stability of the epoxide hydrolase structure (i.e. it can retain proteolytic activity) or maintains a Tm of at least 42oC.
- the fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) can comprise further components as well, including multiple peptides at multiple loops.
- the peptides and nucleic acids encoding them are randomized, either fully randomized or they are biased in their randomization, e.g. in nucleotide/residue frequency generally or per position.
- Randomized means that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively.
- the nucleic acids which give rise to the peptides can be chemically synthesized, and thus may inco ⁇ orate any nucleotide at any position. Thus, when the nucleic acids are expressed to form peptides, any amino acid residue may be inco ⁇ orated at any position.
- the synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized nucleic acids.
- the library can provide a sufficiently structurally diverse population of randomized expression products to affect a probabilistically sufficient range of cellular responses to provide one or more cells exhibiting a desired response.
- the invention provides an interaction library large enough so that at least one of its members will have a structure that gives it affinity for some molecule, protein, or other factor whose activity is necessary for completion of a signaling pathway.
- a peptide library of the invention is fully randomized, with no sequence preferences or constants at any position.
- the library is biased, that is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
- the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
- individual residues may be fixed in the random peptide sequence of the insert to create a structural bias.
- the random libraries can be biased to a particular secondary structure by including an appropriate number of residues (beyond the glycine linkers) which prefer the particular secondary structure.
- the bias is towards peptides that interact with known classes of molecules.
- a short region from the HEV-1 envelope cytoplasmic domain has been previously shown to block the action of cellular calmodulin.
- Regions of the Fas cytoplasmic domain which shows homology to the mastoparan toxin from wasps, can be limited to a short peptide region with death-inducing apoptotic or G protein inducing functions.
- a number of molecules or protein domains are suitable as starting points for the generation of biased randomized peptides.
- a large number of small molecule domains are known, that confer a common function, structure or affinity.
- areas of weak amino acid homology may have strong stractural homology.
- Exemplary molecules, domains, and/or corresponding consensus sequences used in the invention include SH-2 domains, SH-3 domains, Pleckstrin, death domains, epoxide hydrolase cleavage/recognition sites, enzyme inhibitors, enzyme substrates, Traf, etc.
- nucleic acid binding proteins containing domains suitable for use in the invention e.g., leucine zipper consensus sequences.
- the invention provides a variety of expression vectors comprising nucleic acids of the invention, including those encoding a fusion protein.
- the expression vectors may be either self-replicating extra chromosomal vectors or vectors which integrate into a host genome.
- these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the fusion protein.
- control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
- the control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site.
- Transcriptional and translational regulatory sequences used in the expression cassettes and vectors of the invention include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
- the regulatory sequences include a promoter and transcriptional start and stop sequences.
- Promoter sequences encode either constitutive or inducible promoters.
- the promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
- the promoters are strong promoters, allowing high expression in cells, particularly mammalian cells, such as the CMV promoter, particularly in combination with a Tet regulatory element.
- the expression vector may comprise additional elements.
- the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
- the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
- the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
- the nucleic acids or vectors of the invention are introduced into the cells for screening, thus, the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid.
- the method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO4 precipitation, liposome fusion, lipofection (e.g., LEPOFECTINTM), electroporation, viral infection, etc.
- the candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfecting such targets are preferred.
- Expression vectors of the invention may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.
- selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
- the epoxide hydrolase enzymes, fragments thereof and nucleic acids that encode the enzymes and fragments can be affixed to a solid support.
- This is often economical and efficient in the use of epoxide hydrolases in industrial processes.
- a consortium or cocktail of epoxide hydrolase enzymes (or active fragments thereof), which are used in a specific chemical reaction can be attached to a solid support and dunked into a process vat. The enzymatic reaction can occur. Then, the solid support can be taken out of the vat, along with the enzymes affixed thereto, for repeated use.
- an isolated nucleic acid of the invention is affixed to a solid support.
- the solid support is selected from the group of a gel, a resin, a polymer, a ceramic, a glass, a microelectrode and any combination thereof.
- solid supports useful in this invention include gels.
- Some examples of gels include Sepharose, gelatin, glutaraldehyde, chitosan-treated glutaraldehyde, albumin-glutaraldehyde, chitosan-Xanthan, toyopearl gel (polymer gel), alginate, alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magnetic agarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEG hydrogel, phosphorylated polyvinyl alcohol (PVA), monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.
- Another solid support useful in the present invention are resins or polymers.
- resins or polymers include cellulose, acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITETM XAD-7, AMBERLITETM XAD-8, AMBERLITETM TRA-94, AMBERLITETM IRC-50, polyvinyl, polyacrylic, polymethacrylate, or any combination thereof
- another type of solid support useful in the present invention is ceramic.
- Some examples include non-porous ceramic, porous ceramic, SiO , Al 2 O 3 .
- Another type of solid support useful in the present invention is glass. Some examples include non-porous glass, porous glass, aminopropyl glass or any combination thereof.
- solid support Another type of solid support that can be used is a microelectrode.
- An example is a polyethyleneimine-coated magnetite.
- Graphitic particles can be used as a solid support.
- Another example of a solid support is a cell, such as a red blood cell.
- Capillary arrays such as the GIGAMATRIXTM, Diversa Co ⁇ oration, San Diego, CA, can be used to in the methods of the invention.
- Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array, including capillary arrays.
- Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention.
- Capillary arrays provide another system for holding and screening samples.
- a sample screening apparatus can include a plurality of capillaries formed into an array of adjacent capillaries, wherein each capillary comprises at least one wall defining a lumen for retaining a sample.
- the apparatus can further include interstitial material disposed between adjacent capillaries in the array, and one or more reference indicia formed within of the interstitial material.
- a capillary for screening a sample wherein the capillary is adapted for being bound in an array of capillaries, can include a first wall defining a lumen for retaining the sample, and a second wall formed of a filtering material, for filtering excitation energy provided to the lumen to excite the sample.
- a polypeptide or nucleic acid e.g., a ligand
- a first component into at least a portion of a capillary of a capillary array.
- Each capillary of the capillary array can comprise at least one wall defining a lumen for retaining the first component.
- An air bubble can be introduced into the capillary behind the first component.
- a second component can be introduced into the capillary, wherein the second component is separated from the first component by the air bubble.
- a sample of interest can be introduced as a first liquid labeled with a detectable particle into a capillary of a capillary array, wherein each capillary of the capillary array comprises at least one wall defining a lumen for retaining the first liquid and the detectable particle, and wherein the at least one wall is coated with a binding material for binding the detectable particle to the at least one wall.
- the method can further include removing the first liquid from the capillary tube, wherein the bound detectable particle is maintained within the capillary, and introducing a second liquid into the capillary tube.
- the capillary array can include a plurality of individual capillaries comprising at least one outer wall defining a lumen.
- the outer wall of the capillary can be one or more walls fused together. Similarly, the wall can define a lumen that is cylindrical, square, hexagonal or any other geometric shape so long as the walls form a lumen for retention of a liquid or sample.
- the capillaries of the capillary array can be held together in close proximity to form a planar structure.
- the capillaries can be bound together, by being fused (e.g., where the capillaries are made of glass), glued, bonded, or clamped side-by-side.
- the capillary array can be formed of any number of individual capillaries, for example, a range from 100 to 4,000,000 capillaries.
- a capillary array can form a micro titer plate having about 100,000 or more individual capillaries bound together. Arrays, or "Biochips"
- Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array.
- Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention.
- a monitored parameter is transcript expression of an epoxide hydrolase gene.
- One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or "biochip.”
- arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention.
- Polypeptide arrays can also be used to simultaneously quantify a plurality of proteins.
- arrays are generically a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts.
- biological molecules e.g., oligonucleotides
- any known array and/or method of making and using arrays can be inco ⁇ orated in whole or in part, or variations thereof, as described, for example, in U.S. Patent Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr.
- the invention provides isolated or recombinant antibodies that specifically bind to an epoxide hydrolase of the invention. These antibodies can be used to isolate, identify or quantify the fluorescent polypeptides of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention or other related epoxide hydrolases.
- the antibodies can be used in immunoprecipitation, staining, immunoaffinity columns, and the like.
- nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of the invention.
- the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased.
- the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.
- Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
- Polypeptides or peptides can be used to generate antibodies which bind specifically to the polypeptides of the invention.
- the resulting antibodies may be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample.
- a protein preparation such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the polypeptides of the invention.
- the antibody is attached to a solid support, such as a bead or other column matrix.
- the protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of the invention. After a wash to remove non-specifically bound proteins, the specifically bound polypeptides are eluted.
- binding may be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding may be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the sample may be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassays, and Western Blots.
- Polyclonal antibodies generated against the polypeptides of the invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to a non-human animal. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies which may bind to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing that polypeptide.
- any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
- kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, polypeptides (e.g., epoxide hydrolases) and/or antibodies of the invention.
- the kits also can contain instructional material teaching the methodologies and industrial uses of the invention, as described herein.
- Measuring Metabolic Parameters [00343]
- the methods of the invention provide whole cell evolution, or whole cell engineering, of a cell to develop a new cell strain having a new phenotype by modifying the genetic composition of the cell, where the genetic composition is modified by addition to the cell of a nucleic acid.
- At least one metabolic parameter of a modified cell is monitored in the cell in a "real time” or “on-line” time frame.
- a plurality of cells such as a cell culture, is monitored in "real time” or “on-line.”
- a plurality of metabolic parameters is monitored in "real time” or “on-line.” Metabolic parameters can be monitored using the fluorescent polypeptides of the invention.
- Metabolic flux analysis is based on a known biochemistry framework.
- a linearly independent metabolic matrix is constructed based on the law of mass conservation and on the pseudo-steady state hypothesis (PSSH) on the intracellular metabolites.
- PSSH pseudo-steady state hypothesis
- Metabolic phenotype relies on the changes of the whole metabolic network within a cell. Metabolic phenotype relies on the change of pathway utilization with respect to environmental conditions, genetic regulation, developmental state and the genotype, etc.
- the dynamic behavior of the cells are analyzed by investigating the pathway utilization. For example, if the glucose supply is increased and the oxygen decreased during the yeast fermentation, the utilization of respiratory pathways will be reduced and/or stopped, and the utilization of the fermentative pathways will dominate.
- the methods of the invention can help determine how to manipulate the fermentation by determining how to change the substrate supply, temperature, use of inducers, etc. to control the physiological state of cells to move along desirable direction.
- the MFA results can also be compared with transcriptome and proteome data to design experiments and protocols for metabolic engineering or gene shuffling, etc.
- any modified or new phenotype can be conferred and detected, including new or improved characteristics in the cell. Any aspect of metabolism or growth can be monitored. Monitoring expression of an mRNA transcript
- the engineered phenotype comprises increasing or decreasing the expression of an mRNA transcript or generating new transcripts in a cell. This increased or decreased expression can be traced by use of a fluorescent polypeptide of the invention.
- mRNA transcripts, or messages also can be detected and quantified by any method known in the art, including, e.g., Northern blots, quantitative amplification reactions, hybridization to arrays, and the like.
- Quantitative amplification reactions include, e.g., quantitative PCR, including, e.g., quantitative reverse transcription polymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or "real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol. 114:313-318; Xia (2001) Transplantation 72:907-914).
- the engineered phenotype is generated by knocking out expression of a homologous gene.
- the gene's coding sequence or one or more transcriptional control elements can be knocked out, e.g., promoters enhancers.
- promoters enhancers e.g., promoters enhancers.
- the engineered phenotype comprises increasing the expression of a homologous gene. This can be effected by knocking out of a negative control element, including a transcriptional regulatory element acting in cis- or trans- , or, mutagenizing a positive control element.
- a negative control element including a transcriptional regulatory element acting in cis- or trans- , or, mutagenizing a positive control element.
- One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array.
- the engineered phenotype comprises increasing or decreasing the expression of a polypeptide or generating new polypeptides in a cell. This increased or decreased expression can be traced by use of an epoxide hydrolase of the mvention.
- Polypeptides, peptides and amino acids also can be detected and quantified by any method known in the art, including, e.g., nuclear magnetic resonance (NMR), spectrophotometry, radiography (protein radiolabeling), electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, various immunological methods, e.g.
- EHs Several assay methods for obtaining EHs can be used. These assay methods include growth-based assays, direct activity-based assays and sequence-based assays. Preferably, to successfully obtain a range of EHs with desirable characteristics, all three of these assay methods may be used complementarily. Growth-based Assays.
- EHs may be discovered if they convert an epoxide substrate to a diol that can be utilized by host bacteria as a carbon source.
- the library cells are grown in minimal media supplemented with this epoxide as the sole carbon source, only those clones harboring active epoxide hydrolases will be able to produce the corresponding diol and to utilize it as a carbon source for growth and proliferation. Over time, these clones will dominate the microbial population, and thus can be readily isolated.
- Appropriate hosts need to be used for the selection experiments.
- an E. coli fucA-disrupted mutant that can use propane diol as carbon source is required for propylene oxide selection.
- These hosts can be generated via targeted mutation of certain genes or transposon (Tn) mutagenesis in a random fashion. The latter strategy is more attractive because it is more convenient and extremely powerful.
- Tn is introduced into E. coli hosts through electroporation, where in vivo transposition leads to random Tn insertion in genomic DNA. This results in an E.
- coli insertion library suitable for screening for desired mutants, such as those that can utilize propane diol as carbon source.
- desired mutants such as those that can utilize propane diol as carbon source.
- propane diol-utilizing mutants Several insertion libraries of different E. coli hosts will be used to screen for propane diol- utilizing mutants. Specifically, the library cells will be plated out on agar plates containing minimal medium with propane diol as the sole carbon source. Upon incubation, propane diol-utilizing clones can be identified because only they will grow and form colonies on the plates.
- Epoxides are known to be toxic to microbes due to alkylation of proteins and nucleic acids.
- concentrations of glycidol on the growth of an E. coli host was evaluated. The results showed that E. coli can tolerate up to 0.05% of glycidol (v/v). This concentration may be high enough for selection as the cells were able to grow with 0.025% glycerol provided extracellularly in the media as the sole carbon source. If necessary, however, E. coli mutants bearing higher glycidol tolerance may be discovered by screening libraries of mutagenized hosts including the Tn insertion libraries mentioned above.
- a positive control clone has been developed that has epoxide hydrolase activity. Having such a control is useful because it can be used to guide and evaluate the assay development for both the selection and screening.
- An epoxide hydrolase from A. radiobacter whose nucleotide sequence was reported, can be readily cloned and expressed in E. coli. (Arand, M.; Oesch, F. Biochem. J. 1999, 344, 273-280.) Primers have been designed and synthesized for the amplification and cloning of this gene.
- an active epoxide hydrolase that may be used as the positive control has been identified. Sequence based Assays
- a complementary approach to the activity-based discovery of epoxide hydrolases is sequence-based discovery of epoxide hydrolases followed by assessment of their substrate specificities in secondary assays. Using sequence-based methods is a valuable strategy for discovering particular classes of enzymes. Considerable amounts of sequence and stractural information are available on EHs, rendering the development of sequenced-based discovery possible.
- Bacterial EHs have higher sequence similarity. Alignments of the nucleotide sequences of bacterial EHs will allow the identification of conserved sequences. Primers will be designed based on these regions. These primers will be used to generate PCR products from DNA libraries. The products will be gel-separated, purified, and subjected for sequence analysis. The full-length sequences of positive hits can be retrieved by southern blotting. The activities of these hits will then be investigated using fluorogenic or chromogemc assays.
- sequence-based approach compared to activity-based methods is that it is limited to the discovery of genes that share homology to existing genes.
- sequence-based approach becomes increasingly powerful as more sequences can be used in probe design.
- Bioinformatic analysis of a DNA database resulted in a total of 6 putative epoxide hydrolase genes as well as 3 partial open reading frame (ORFs) that bear homology to A. radiobacter and other epoxide hydrolases. Based on the conserved nucleotide sequences extracted from these ORFs, degenerate primers have been designed and used for screening a gene library known to contain one of these genes.
- Fluorogenic and chromogenic assays have been used to great effect in high- throughput screening for enzyme characterization and discovery. Fluorogenic assays have been commonly used for many hydrolytic enzymes in which the substrates release a fluorescent signal upon the hydrolysis reaction. These assays are activity-based like the selection method, but they can be used for more diverse substrates than the selection experiments. The limitation, however, is that they have lower throughput than the selection assays.
- the epoxide substrate (13) used in this assay contains a masked fluorophore that can generate strong fluorescence when released.
- periodate is added to oxidize the vicinal diol product (14) to generate a carbonyl-containing intermediate (15).
- 15 can undergo a b-elimination reaction catalyzed by bovine serum albumin (BSA) to release a fluorescent product (16) such as umbelliferone.
- BSA bovine serum albumin
- the assay is carried out in a 1536-well format. Clones from gene libraries are distributed into individual wells, preferably 5 clones per well for the primary screening. These clones are allowed to grow for 24-48 hrs before substrate 19 is added. After 2 hours of incubation, sodium periodate and BSA are added to promote the b-elimination reaction. The fluorescence level in each well is measured to identify preliminary hits. These hits can be reconfirmed by running a second round of assays. Robotic systems have been developed to automate all the liquid handling and fluorescence measurement processes.
- the first substrate for this assay development, 19, has been synthesized according to Figure 17. Coupling of umbelliferone (17) with 4-bromo-l-butene in the presence of potassium carbonate at 50 °C yielded olefin 18, which was subjected to an epoxidation reaction using meta-chloroperbenzoic acid (mCPBA). The resulting epoxide 19 was used to detect epoxide hydrolase activity of the 6 clones mentioned above. These clones contain putative epoxide hydrolase genes. One of them was found to be active for 19. This showed that the assay is useful. Colorimetric Assay
- a colorimetric assay can be extremely useful in high throughput screening if a sensitive color change is involved and the assay can be performed in solid agar format. Screening on solid agar offers extremely high throughput, while a color change allows easy identification of hits.
- Hydrolyzed epoxides are not reactive with 20, and thus observation of a decrease in absorbance at 560 nm is indicative of epoxide hydrolysis.
- colonies grown on agar plates were transferred to filter paper preincubated with epoxides.
- Epoxide hydrolase activity was detected by the formation of colorless halos on the blue filter paper.
- This assay has the potential to be converted to a HTP screen.
- the disadvantage of this assay is that it detects the disappearance of the substrate instead of the appearance of the products.
- the advantage is that it targets substrates directly, not their derivatives. Therefore, even if its relative low sensitivity proves to be a problem for HTP screening, it maybe used for secondary screening of primary hits detected from other discovery methods.
- the assays developed will be applied to screen the environmental gene libraries for the presence of microbial enzymes with the necessary activities and substrate specificities. Positive hits from these screens may then be sequenced and the genes subcloned into expression vectors. The expressed recombinant enzymes can then be characterized with respect to activity and substrate selectivities. Should the identified enzymes require enhancement of one or more of their properties (e.g. pH and temperature optima, thermostability, thermotolerance, substrate specificity etc.) they can be optimized using GSSMTM (Gene Site Saturation Mutagenesis), Gene ReassemblyTM and other technologies discussed below.
- GSSMTM Gene Site Saturation Mutagenesis
- epoxide hydrolases may be used in the chemo-enzymatic synthesis of specific fine chemicals and high value precursors to pharmaceuticals and agrochemicals.
- the optimized enzymes developed using a method of the present invention may be applied in the development of a commercially viable synthesis route to one or more target compounds.
- the epoxide hydrolases can be used as key intermediates in the synthesis of fine chemicals and enantiomeric pharmaceuticals having the desired purities.
- the environmental gene libraries are constructed using DNA isolated from a wide variety of micro-environments around the world. Application of an appropriate discovery method then allows enzymes to be extracted from these libraries according to function, enzyme class or a specific combination of the two. In contrast to traditional discovery programs, the preferred discovery method ensures capture of genes from uncultivated microbes and facilitates screening in well-defined, domesticated laboratory hosts. This expression cloning method results in simultaneous capture of enzyme activities and the corresponding genetic information.
- a discovery method involves: isolating and fractionating nucleic acids from nature or other suitable sources; constructing environmental gene libraries; screening the genes in the environmental libraries to discover the desired genes encoding the desired enzymes using the methods described below; optimizing the desired genes to optimize the activity of the desired enzymes using the evolution technologies described in U.S. Patent No. 5,830,696, U.S. Patent No. 5,939,250 and U.S. Patent No. 5,965,408, which are inco ⁇ orated herein by reference; sequencing the optimized genes; overexpressing the sequenced genes in suitable host strains; producing a large number of the suitable strains containing the optimized genes by fermentation and obtaining the desired enzymes, optionally contained in host strains, after purification.
- Newly cloned or discovered enzymes can then be further customized by using the evolution technologies described in U.S. Patent No. 5,830,696, U.S. Patent No. 5,939,250 and U.S. Patent No. 5,965,408 and a combinatorial evolution technology described below.
- the screening step in one aspect of the present invention may be carried out by one or more of expression and sequence-based screening methods including single cell activity screens, microtiter plate-based activity screens, sequence-based screening and growth selection methods. These methods may all be applied to the discovery of epoxide hydrolases utilizing the assays described above.
- Single cell activity screening method is a method derived from Fluorescence Activated Cell Sorting (FACS) by substantially modifying the FACS platform for expression and sequence hybridization-based screening of environmental libraries ( Figure 18).
- FACS-hybridization cloning methodology permits the recovery of recombinant clones based on sequence homology.
- This single cell activity screening method allows screening rates of 50,000 clones per second and a daily screening rate of up to 109 clones.
- the growth selection method can be one of the most powerful methods for enzyme discovery. In this method the substrate of choice acts as a nutrient source for the host cells only when those cells contain the enzyme activity of interest, allowing them to grow selectively.
- sequence-based discovery methods may be powerful and complementary alternatives to expression cloning.
- Both solution phase and FACS-based formats can be used for ultra high throughput DNA hybridization-based discovery techniques, such as environmental biopanning, which facilitate screening of the large and complex environmental gene libraries.
- inserts from mega libraries are rendered single stranded and combined in solution with arrays of biotinylated hybridization probes known as hooks ( Figure 19).
- Hooks Figure 19
- the eluted sequence-enriched DNA inserts are then either subjected to another cycle of biopanning or back-cloned into lambda. In this way enrichment is achieved greater than 1000-fold for sequences of interest.
- the FACS-based biopanning approach further facilitates the enzyme identification process by allowing for amplification-free biopanning of both small and large insert clones.
- Laboratory evolution of enzymes can be used to further improve, customize or refine the properties of the enzymes. These laboratory evolution technologies include Gene Site Saturation Mutagenesis (GSSMTM) and GeneReassemblyTM, where multiple natural genes can be combined to create a combinatorial evolution library. If necessary, these technologies can be applied to the epoxide hydrolases discovered using the enzyme discovery method to further optimize these epoxide hydrolases for characteristics such as thermostability, specific activity or stereospecificity.
- the present invention provides rapid screening of libraries derived from more than one organism, such as a mixed population of organisms from, for example, an environmental sample or an uncultivated population of organisms or a cultivated population of organisms.
- gene libraries are generated by obtaining nucleic acids from a mixed population of organisms and cloning the nucleic acids into a suitable vector for transforming a plurality of clones to generate a gene library.
- the gene library thus contains gene or gene fragments present in organisms of the mixed population.
- the gene library can be an expression library, in which case the library can be screened for an expressed polypeptide having a desired activity.
- the gene library can be screened for sequences of interest by, for example, PCR or hybridization screening.
- nucleic acids from isolates of a sample containing a mixed population of organism are pooled and the pooled nucleic acids are used to generate a gene library.
- Isolates is meant that a particular species, genus, family, order, or class of organisms is obtained or derived from a sample having more than one organism or from a mixed population of organisms. Nucleic acids from these isolated populations can then be used to generate a gene library. Isolates can be obtained from by selectively filtering or culturing a sample containing more than one organism or a mixed population of organisms. For example, isolates of bacteria can be obtained by filtering the sample through a filter, which excludes organisms based on size or by culturing the sample on media that allows from selective growth or selective inhibition of certain populations of organisms.
- An "enriched population” is a population of organisms wherein the percentage of organisms belonging to a particular species, genus, family, order or class of organisms is increased with respect to the population as a whole.
- selective growth or inhibition media can increase the overall number of organisms.
- One can enrich for prokaryotic organisms with respect to the total number of organisms in the population.
- a particular species, genus, family, order or class of organisms can be enriched by growing a mixed population on a selective media that inhibits or promotes the growth of a subpopulation within the mixed population.
- nucleic acids from a plurality (e.g., two or more) of isolates from a mixed population of organisms are used to generate a plurality of gene libraries containing a plurality of clones, and the gene libraries from at least two isolates are then pooled to obtain a "pooled isolate library.”
- the clones are screened to detect a bioactivity, in this case activity as a catalyst for the modification of epoxides or a biomolecule of interest (e.g., an EH).
- a bioactivity in this case activity as a catalyst for the modification of epoxides or a biomolecule of interest (e.g., an EH).
- screening techniques include, for example, contacting a clone, clonal population, or population of nucleic acid sequences with a substrate or substrates having a detectable molecule that provides a detectable signal upon interaction with the bioactivity or biomolecule of interest.
- the substrate can be an enzymatic substrate, a bioactive molecule, an oligonucleotide, and the like.
- gene libraries are generated, clones are either exposed to a chromagemc or fluorogenic substrate or substrate(s) of interest, or hybridized to a labeled probe (e.g., an oligonucleotide having a detectable molecule) having a sequence corresponding to a sequence of interest and positive clones are identified by a detectable signal (e.g., fluorescence emission).
- a labeled probe e.g., an oligonucleotide having a detectable molecule
- expression libraries generated from a mixed population of organisms are screened for an activity of interest. Specifically, expression libraries are generated, clones are exposed to the substrate or substrate(s) of interest, and positive clone are identified and isolated.
- the present invention does not require cells to survive. The cells only need to be viable long enough to produce the molecule to be detected, and can thereafter be either viable or nonviable cells, so long as the expressed biomolecule (e.g., an enzyme) remains active.
- the invention provides an approach that combines direct cloning of genes encoding novel or desired bioactivities from environmental samples with a high-throughput screening system designed for the rapid discovery of new molecules, for example, enzymes.
- the approach is based on the construction of environmental "expression libraries" which can represent the collective genomes of numerous naturally occurring microorganisms archived in cloning vectors that can be propagated in E. coli or other suitable host cells. Because the cloned DNA can be initially extracted directly from environmental samples or from isolates of the environmental samples, the libraries are not limit o the small fraction of prokaryotes that can be grown in pure culture.
- a normalization of the environmental DNA present in these samples could allow a more equal representation of the DNA from all of the species present in a sample. Normalization techniques (described below) can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample that may be under-represented by several orders of magnitude compared to the dominant species in the sample. Normalization can occur in any of the foregoing embodiments following obtaining nucleic acids from the sample or isolate(s).
- the invention provides a high-throughput capillary array system for screemng that allows one to assess an enormous number of clones to identify and recover cells encoding useful enzymes, as well as other biomolecules (e.g., ligands).
- the capillary array-based techniques described herein can be used to screen, identify and recover proteins having a desired bioactivity or other ligands having a desired binding affinity.
- binding assays may be conducted by using an appropriate substrate or other marker that emits a detectable signal upon the occurrence of the desired binding event.
- fluorescence activated cell sorting can be used to screen and isolate clones having an activity or sequence of interest.
- FACS machines have been employed in the studies focused on the analyses of eukaryotic and prokaryotic cell lines and cell culture processes.
- FACS has also been utilized to monitor production of foreign proteins in both eukaryotes and prokaryotes to study, for example, differential gene expression, and the like.
- the detection and counting capabilities of the FACS system have been applied in these examples.
- FACS has never previously been employed in a discovery process to screen for and recover bioactivities in prokaryotes.
- the present invention does not require cells to survive, as do previously described technologies, since the desired nucleic acid (recombinant clones) can be obtained from alive or dead cells.
- the cells only need to be viable long enough to produce the compound to be detected, and can thereafter be either viable or non-viable cells so long as the expressed biomolecule remains active.
- the present invention also solves problems that would have been associated with detection and sorting ofE. coli expressing recombinant enzymes, and recovering encoding nucleic acids.
- the present invention includes within its embodiments any apparatus capable of detecting fluorescent wavelengths associated with biological material, such apparatus are defined herein as fluorescent analyzers (one example of which is a FACS apparatus).
- nucleic acid sequences of interest can be identified or "biopanned" by contacting a clone, device (e.g. a gene chip), filter, or nucleic acid sample with a probe labeled with a detectable molecule.
- the probe will typically have a sequence that is substantially identical to the nucleic acid sequence of interest.
- the probe will be a fragment or full length nucleic acid sequence encoding a polypeptide of interest.
- the probe and nucleic acids are incubated under conditions and for such time as to allow the probe and a substantially complementary sequence to hybridize.
- Hybridization stringency will vary depending on, for example, the length and GC content of the probe. Such factors can be determined empirically (See, for example, Sambrook et al., Molecular Cloning ⁇ A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, M. Ausubel et al., eds., (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., most recent Supplement)).
- the complementary sequence can be PCR amplified, identified by hybridization techniques (e.g., exposing the probe and nucleic acid mixture to a film), or detecting the nucleic acid using a chip.
- the present invention allows the rapid screening of complex environmental libraries, containing, for example, genomic sequences from thousands of different organisms or subsets and isolates thereof.
- the benefits of the present invention can be seen, for example, in screening a complex environmental sample. Screening of a complex sample previously required one to use labor-intensive methods to screen several million clones to cover the genomic biodiversity.
- the invention represents an extremely high-throughput screening method, which allows one to assess this enormous number of clones.
- the method disclosed allows the screening anywhere from about 30 million to about 200 million clones per hour for a desired nucleic acid sequence, biological activity, or biomolecule of interest. This allows the thorough screening of environmental libraries for clones expressing novel bioactivities or biomolecules.
- sequence or bioactivity of interest e.g., an enzyme of interest
- the sequence or polynucleotide encoding the bioactivity of interest can be evolved, mutated or derived to modify the amino acid sequence to provide, for example, modified activities such as increased thermostability, specificity or activity.
- the invention provides methods of identifying a nucleic acid sequence encoding a polypeptide having either known or unknown function. For example, much of the diversity in microbial genomes results from the rearrangement of gene clusters in the genome of microorganisms. These gene clusters can be present across species or phylogenetically related with other organisms.
- genes and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes.
- the genes are clustered, in stractures referred to as "gene clusters,” on a single chromosome and are transcribed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster.
- the gene cluster, the promoter, and additional sequences that function in regulation altogether are referred to as an "operon" and can include up to 20 or more genes, usually from 2 to 6 genes.
- a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function.
- Clustering is a prerequisite for maintaining identity between genes, although clustered genes are not necessarily identical. Gene clusters range from extremes where a duplication is generated to adjacent related genes to cases where hundreds of identical genes lie in a tandem array. Sometimes no significance is discemable in a repetition of a particular gene. A principal example of this is the expressed duplicate insulin genes in some species, whereas a single insulin gene is adequate in other mammalian species.
- gene clusters undergo continual reorganization and, thus, the ability to create heterogeneous libraries of gene clusters from, for example, bacterial or other prokaryote sources is valuable in determining sources of novel proteins, particularly including enzymes such as, for example, the polyketide synthases that are responsible for the synthesis of polyketides having a vast array of useful activities.
- polyketides are molecules which are an extremely rich source of bioactivities, including antibiotics (such as tetracyclines and erythromycin), anti-cancer agents (daunomycin), immunosuppressants (FK506 and rapamycin), and veterinary products (monensin).
- Polyketides produced by polyketide synthases are valuable as therapeutic agents.
- Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in length and patterns of functionality and cyclization.
- Polyketide synthase genes fall into gene clusters and at least one type (designated type I) of polyketide synthases have large size genes and enzymes, complicating genetic manipulation and in vitro studies of these genes/proteins.
- Other types of proteins that are the product(s) of gene clusters are also contemplated, including, for example, antibiotics, antivirals, antitumor agents and regulatory proteins, such as insulin.
- a gene cluster can be ligated into a vector containing an expression of regulatory sequences, which can control and regulate the production of a detectable protein or protein-related array activity from the ligated gene clusters.
- vectors which have an exceptionally large capacity for exogenous nucleic acid introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) ofE. coli.
- This f- factor ofE. coli is a plasmid which affects high-frequency transfer of itself during conjugation and is ideal to achieve and stably propagate large nucleic acid fragments, such as gene clusters from mixed microbial samples.
- the nucleic acid isolated or derived from these samples e.g., a mixed population of microorganisms
- Such vectors or plasmids are typically those containing expression regulatory sequences, including promoters, enhancers and the like.
- the invention provides novel systems to clone and screen mixed populations of organisms, enriched samples, or isolates thereof for polynucleotides encoding molecules having an activity of interest, enzymatic activities and bioactivities of interest in vitro.
- the method(s) of the invention allow the cloning and discovery of novel bioactive molecules in vitro, and in particular novel bioactive molecules derived from uncultivated or cultivated samples. Large size gene clusters, genes and gene fragments can be cloned, sequenced and screened using the method(s) of the invention.
- the method(s) of the invention allow one to clone screen and identify polynucleotides and the polypeptides encoded by these polynucleotides in vitro from a wide range of environmental samples.
- the invention allows one to screen for and identify polynucleotide sequences from complex environmental samples, enriched samples thereof, or isolates thereof.
- Gene libraries can be generated from cell free samples, so long as the sample contains nucleic acid sequences, or from samples containing cells, cellular material or viral particles.
- the organisms from which the libraries may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, lower eukaryotic microorganisms such as fungi, algae and protozoa, as well as mixed populations of plants, plant spores and pollen.
- the organisms may be cultured organisms or uncultured organisms, obtained from environmental samples and includes extremophiles, such as thermophiles, hyperthermophiles, psychrophiles and psychrotrophs.
- Sources of nucleic acids used to generate a DNA library can be obtained from environmental samples, such as, but not limited to, microbial samples obtained from Arctic and Antarctic ice, water or permafrost sources, materials of volcanic origin, materials from soil or plant sources in tropical areas, droppings from various organisms including mammals and invertebrates, as well as dead and decaying matter and the like.
- the nucleic acids used to generate the gene libraries can be obtained, for example, from enriched subpopulations or isolates of the sample.
- DNA of a plurality of isolates can be pooled to create a source of nucleic acids for generation of the library.
- the nucleic acids can be obtained from a plurality of isolates, a plurality of gene libraries generated from the plurality of isolates to obtain a plurality of gene libraries. Two or more of the gene libraries can be pooled or combined to obtain a pooled isolate library.
- nucleic acids may be recovered from either a cultured or non-cultured organism and used to produce an appropriate gene library (e.g., a recombinant expression library) for subsequent determination of the identity of the particular biomolecule of interest (e.g., a polynucleotide sequence) or screened for a bioactivity of interest (e.g., an enzyme or biological activity).
- an appropriate gene library e.g., a recombinant expression library
- the identity of the particular biomolecule of interest e.g., a polynucleotide sequence
- a bioactivity of interest e.g., an enzyme or biological activity
- an environmental sample is any sample containing organisms or polynucleotides or a combination thereof.
- an environmental sample can be obtained from any number of sources (as described above), including, for example, insect feces, hot springs, soil and the like. Any source of nucleic acids in purified or non- purified form can be utilized as starting material. Thus, the nucleic acids may be obtained from any source, which is contaminated by an organism or from any sample containing cells.
- the environmental sample can be an extract from any bodily sample such as blood, urine, spinal fluid, tissue, vaginal swab, stool, amniotic fluid or buccal mouthwash from any mammalian organism.
- sample can be a tissue sample, salivary sample, fecal material or material in the digestive tract of the organism.
- An environmental sample also includes samples obtained from extreme environments including, for example, hot sulfur pools, volcanic vents, and frozen tundra. The sample can come from a variety of sources.
- the sample in horticulture and agricultural testing can be a plant, fertilizer, soil, liquid or other horticultural or agricultural product; in food testing the sample can be fresh food or processed food (for example infant formula, seafood, fresh produce and packaged food); and in environmental testing the sample can be liquid, soil, sewage treatment, sludge and any other sample in the environment which is considered or suspected of containing an organism or polynucleotides.
- the sample is a mixture of material containing a mixed population of organisms, for example, blood, soil or sludge
- a mixed population of organisms for example, blood, soil or sludge
- it can be treated with an appropriate reagent which is effective to open the cells and expose or separate the strands of nucleic acids.
- this lysing and nucleic acid denaturing step will allow cloning, amplification or sequencing to occur more readily.
- the mixed population can be cultured prior to analysis in order to purify or enrich a particular population or a desired isolate (e.g., an isolate of a particular species, genus, or family of organisms) and thus obtaining a purer sample. This is not necessary, however.
- culturing of organisms in the sample can include culturing the organisms in microdroplets and separating the cultured microdroplets with a cell sorter into individual wells of a multi-well tissue culture plate.
- the sample can be cultured on any number of selective media compositions designed to inhibit or promote growth of a particular subpopulation of organisms.
- nucleic acids can be obtained from the isolates as described below.
- the nucleic acids obtained from the isolates can be used to generate a gene library or, alternatively, be pooled with other isolate fractions of the sample wherein the pooled nucleic acids are used to generate a gene library.
- the isolates can be cultured prior to extraction of nucleic acids or can be uncultured. Methods of isolating specific populations of organisms present in a mixed population.
- the sample comprises nucleic acids from, for example, a diverse and mixed population of organisms (e.g., microorganisms present in the gut of an insect). Nucleic acids are isolated from the sample using any number of methods for DNA and RNA isolation. Such nucleic acid isolation methods are commonly performed in the art. Where the nucleic acid is RNA, the RNA can be reversed transcribed to DNA using primers known in the art. Where the DNA is genomic DNA, the DNA can be sheared using, for example, a 25 -gauge needle. [00406] The nucleic acids can be cloned into an appropriate vector.
- the vector used will depend upon whether the DNA is to be expressed, amplified, sequenced or manipulated in any number of ways known in the art (see, for example, U.S. Patent No. 6,022,716 which discloses high throughput sequencing vectors). Cloning techniques are known in the art or can be developed by one skilled in the art, without undue experimentation. The choice of a vector will also depend on the size of the polynucleotide sequence and the host cell to be employed in the methods of the invention.
- the vector used in the invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzaviras, he ⁇ esvirases, reovirases, paramyxovirases, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein).
- viruses e.g., retroviruses, parainfluenzaviras, he ⁇ esvirases, reovirases, paramyxovirases, and the like
- selected portions thereof e.g., coat protein, spike glycoprotein, capsid protein.
- cosmids and phagemids are typically used where the specific nucleic acid sequence to be analyzed or modified is large because these vectors are able to stably propagate large polynucleotides.
- the vector containing the cloned nucleic acid sequence can then be amplified by plating (i.e., clonal amplification) or transfecting a suitable host cell with the vector (e.g., a phage on an E. coli host).
- the cloned nucleic acid sequence is used to prepare a library for screening (e.g., expression screening, PCR screening, hybridization screening or the like) by transforming a suitable organism.
- Hosts known in the art are transformed by artificial introduction of the vectors containing the nucleic acid sequence by inoculation under conditions conducive for such transformation.
- transform or transformation is meant a permanent or transient genetic change induced in a cell following inco ⁇ oration of new DNA (e.g., DNA exogenous to the cell).
- new DNA e.g., DNA exogenous to the cell
- a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
- a transformed cell or host cell generally refers to a cell (e.g., prokaryotic or eukaryotic) into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule not normally present in the host organism.
- a particular type of vector for use in the invention contains an f-factor origin replication.
- the f-factor (or fertility factor) in E. coli is a plasmid which effects high- frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself.
- cloning vectors referred to as "fosmids" or bacterial artificial chromosome (BAC) vectors are used. These are derived from E. coli f-factor which is able to stably integrate large segments of DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable environmental gene library.
- nucleic acids derived from a mixed population or sample may be inserted into the vector by a variety of procedures.
- the nucleic acid sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
- a typical cloning scenario may have DNA "blunted" with an appropriate nuclease (e.g., Mung Bean Nuclease), methylated with, for example, EcoR I Methylase and ligated to EcoR I linkers GGAATTCC (SEQ ED NO: 1).
- the linkers are then digested with an EcoR I Restriction Endonuclease and the DNA size fractionated (e.g., using a sucrose gradient).
- the resulting size fractionated DNA is then ligated into a suitable vector for sequencing, screening or expression (e.g., a lambda vector and packaged using an in vitro lambda packaging extract).
- Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
- the host is prokaryotic, such as E. coli
- competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC12 method by procedures well known in the art.
- CaC12 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
- Eukaryotic cells can also be cofransfected with a second foreign DNA molecule encoding a selectable marker, such as the he ⁇ es simplex thymidine kinase gene.
- a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
- SV40 simian virus 40
- bovine papilloma virus bovine papilloma virus
- the eukaryotic cell may be a yeast cell (e.g., Saccharomyces cerevisiae), an insect cell (e.g., Drosophila sp.) or may be a mammalian cell, including a human cell.
- Eukaryotic systems and mammalian expression systems, allow for post- translational modifications of expressed mammalian proteins to occur.
- Eukaryotic cells which possess the cellular machinery for processing of the primary transcript, glycosylation, phosphorylation, or secretion of the gene product should be used.
- host cell lines may include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.
- the clones are resuspended in a liquid media, for example, a nutrient rich broth or other growth media known in the art.
- a liquid media for example, a nutrient rich broth or other growth media known in the art.
- the media is a liquid media, which can be readily pipetted.
- One or more media types containing at least one clone of the library are then introduced either individually or together as a mixture, into capillaries (all or a portion thereof) in a capillary array.
- the library is first biopanned prior to introduction or delivery into a capillary device or other screening technique. Such biopanning methods enrich the library for sequences or activities of interest. Examples of methods for biopanning or enrichment are described below.
- the library can be screened or sorted to enrich for clones containing a sequence or activity of interested based on polynucleotide sequences present in the library or clone.
- the invention provides methods and compositions useful in screening organisms for a desired biological activity or biological sequence and to assist in obtaining sequences of interest that can further be used in directed evolution, molecular biology, biotechnological and industrial applications.
- the invention provides methods to rapidly screen, enrich and/or identify sequences in a sample by screening and identifying the nucleic acid sequences present in the sample.
- the invention increases the repertoire of available sequences that can be used for the development of diagnostics, therapeutics or molecules for industrial applications. Accordingly, the methods of the invention can identify novel nucleic acid sequences encoding proteins or polypeptides having a desired biological activity.
- the “biopanning” procedure refers to a process for identifying clones having a specified biological activity by screening for sequence homology in a library of clones.
- the probe sequence used for selectively interacting with the target sequence of interest in the library can be a full-length coding region sequence or a partial coding region sequence for a known bioactivity.
- the library can be probed using mixtures of probes comprising at least a portion of the sequence encoding a known bioactivity or having a desired bioactivity. These probes or probe libraries are preferably single- stranded.
- the library is preferably been converted into single-stranded form.
- the probes that are particularly suitable are those derived from DNA encoding bioactivities having an activity similar or identical to the specified bioactivity, which is to be screened.
- the probes can be used to PCR amplify and thus select target sequences.
- the probe sequences can be used as hybridization probes which can be used to identify sequences with substantial or a desired homology.
- in vivo biopanning may be performed utilizing a FACS- based machine.
- Gene libraries or expression libraries are constructed with vectors, which contain elements, which stabilize transcribed RNA.
- the probe molecules used in the biopanning process consist of oligonucleotides labeled with detectable molecules that provide a detectable signal upon interaction with a target sequence (e.g., only fluoresce upon binding of the probe to a target molecule).
- detectable molecules e.g., only fluoresce upon binding of the probe to a target molecule.
- Various dyes or stains well known in the art for example those described in "Practical Flow Cytometry", 1995 Wiley-Liss, Inc., Howard M.
- Shapiro, M.D. can be used to intercalate or associate with nucleic acid in order to "label" the oligonucleotides.
- These probes are introduced into the recombinant cells of the library using one of several transformation methods.
- the probe molecules interact or hybridize to the transcribed target mRNA or DNA resulting in DNA/RNA heteroduplex molecules or DNA/DNA duplex molecules. Binding of the probe to a target will yield a detectable signal (e.g., a fluorescent signal), which is detected and sorted by a FACS machine, or the like, during the screening process.
- a detectable signal e.g., a fluorescent signal
- the probe DNA should be at least about 10 bases and preferably at least 15 bases. In one aspect, an entire coding region of one part of a pathway may be employed as a probe. Where the probe is hybridized to the target DNA in an in vitro system, conditions for the hybridization in which target DNA is selectively isolated by the use of at least one DNA probe will be designed to provide a hybridization stringency of at least about 50% sequence identity, more particularly a stringency providing for a sequence identity of at least about 70%.
- the resultant libraries of transformed clones can then be further screened for clones, which display an activity of interest.
- Clones can be shuttled in alternative hosts for expression of active compounds, or screened using methods described herein.
- An alternative to the in vivo biopanning described above is an encapsulation technique such as, for example, gel microdroplets, which may be employed to localize multiple clones in one location to be screened on a FACS machine. Clones can then be broken out into individual clones to be screened again on a FACS machine to identify positive individual clones. Screening in this manner using a FACS machine is fully described in patent application Ser. No. 08/876,276 filed Jun. 16, 1997. Thus, for example, if a clone mixture has a desirable activity, then the individual clones may be recovered and rescreened utilizing a FACS machine to determine which of such clones has the specified desirable activity.
- encapsulation strategies and compounds or polymers can be used with the present invention.
- high temperature agarose can be employed for making microdroplets stable at high temperatures, allowing stable encapsulation of cells subsequent to heat-kill steps utilized to remove all background activities when screening for thermostable bioactivities.
- Encapsulation can be in beads, high temperature agaroses, gel microdroplets, cells, such as ghost red blood cells or macrophages, liposomes, or any other means of encapsulating and localizing molecules.
- methods of preparing liposomes have been described (e.g., U.S.
- Patent No.'s 5,653,996, 5393530 and 5,651,981) as well as the use of liposomes to encapsulate a variety of molecules (e.g., U.S. Patent Nos. 5,595,756, 5,605,703, 5,627,159, 5,652,225, 5,567,433, 4,235,871, 5,227,170). Entrapment of proteins, virases, bacteria and DNA in erythrocytes during endocytosis has been described, as well (see, for example, Journal of Applied Biochemistry 4, 418D435 (1982)).
- Microenvironment is any molecular structure, which provides an appropriate environment for facilitating the interactions necessary for the method of the invention.
- An environment suitable for facilitating molecular interactions includes, for example, liposomes.
- Liposomes can be prepared from a variety of lipids including phospholipids, glycolipids, steroids, long-chain alkyl esters; e.g., alkyl phosphates, fatty acid esters; e.g., lecithin, fatty amines and the like.
- a mixture of fatty material may be employed such a combination of neutral steroid, a charge amphiphile and a phospholipid.
- Illustrative examples of phospholipids include lecithin, sphingomyelin and dipalmitoylphos-phatidylcholine.
- Representative steroids include cholesterol, cholestanol and lanosterol.
- Representative charged amphiphilic compounds generally contain from 12-30 carbon atoms.
- Exemplary compounds include mono- or dialkyl phosphate esters, or alkyl amines; e.g., diacetyl phosphate, stearyl amine, hexadecyl amine, dilauryl phosphate, and the like.
- a normalization step is performed prior to generation of the expression library, the expression library is then generated, the expression library so generated is then biopanned, and the biopanned expression library is then screened using a high throughput cell sorting and screening instrument.
- a normalization step is performed prior to generation of the expression library, the expression library is then generated, the expression library so generated is then biopanned, and the biopanned expression library is then screened using a high throughput cell sorting and screening instrument.
- the nucleic acids used to generate a library can be obtained, for example, from environmental samples, mixed populations of organisms (e.g., cultured or uncultured), enriched populations thereof, and isolates thereof.
- the screening techmques include, for example, hybridization screening, PCR screening, expression screening, and the like.
- the gel microdroplet technology has had significance in amplifying the signals available in flow cytometric analysis, and in permitting the screening of microbial strains in strain improvement programs for biotechnology.
- Wittrap et al. (Biotechnolo. Bioeng. (1993) 42:351-356) developed a microencapsulation selection method which allows the rapid and quantitative screening of > 106 yeast cells for enhanced secretion of Aspergillus awamori glucoamylase. The method provides a 400-fold single-pass enrichment for high-secretion mutants.
- Gel microdroplet or other related technologies can be used in the present invention to localize, sort as well as amplify signals in the high throughput screening of recombinant libraries. Cell viability during the screening is not an issue or concern since nucleic acid can be recovered from the microdroplet.
- the enriched clones are suspended in a liquid media such as a nutrient broth or other growth media.
- a liquid media such as a nutrient broth or other growth media.
- the enriched clones comprise a plurality of host cells transformed with constructs comprising vectors into which have been inco ⁇ orated nucleic acid sequences derived from a sample (e.g., mixed populations of organisms, isolates thereof, and the like).
- Liquid media containing a subset of clones and one or more substrates having a detectable molecule is then introduced or contacted, either individually or together as a mixture, with the enriched clones (e.g., into capillaries in a capillary array).
- Interaction (including reaction) of the substrate and a clone expressing an enzyme having the desire enzyme activity produces a product or a detectable signal, which can be spatially detected to identify one or more clones or capillaries containing at least one signal-producing clone.
- the signal-producing clones or nucleic acids contained in the signal-producing clone can then be recovered using any number of techniques.
- a "substrate” as used herein includes, for example, substrates for the detection of a bioactivity or biomolecule (e.g., an enzymes and their specific enzyme activities). Such substrates are well known in the art. For example, various enzymes and suitable substrates specific for such enzymes are provided in Molecular Probes, Handbook Of Fluorescent Probes and Research Chemical (Molecular Probes, Inc.; Eugene, OR), the disclosure of which is inco ⁇ orated herein by reference.
- the substrate can have a detectable molecule associated with it including, for example, chromagemc or fluorogenic molecules.
- a suitable substrate for use in the present invention is any substrate that produces an optically detectable signal upon interaction (e.g., reaction) with a given enzyme having a desired activity, or a given clone encoding such enzyme.
- a suitable substrate based on a desired enzyme activity, for example. Examples of desired enzymes/enzymatic activities include those listed herein.
- a desired enzyme activity may also comprise a group of enzymes in an enzymatic pathway for wliich there exists an optical signal substrate. One example of this is the set of carotenoid synthesis enzymes.
- Substrates are known and/or are commercially available for glycosidases, epoxide hydrolases, phosphatases, and monoxygenases, among others. Where the desired activity is in the same class as that of other biomolecules or enzymes having a number of known subsfrates, the activity can be examined using a cocktail of the known substrates. For example, substrates are known for approximately 20 commercially available esterases and the combination of these known substrates can provide detectable, if not optimal, signal production.
- the optical signal substrate can be a chromogenic substrate, a fluorogenic substrate, a bio-or chemi-luminescent substrate, or a fluorescence resonance energy transfer (FRET) substrate.
- the detectable species can be one, which results from cleavage of the substrate or a secondary molecule which is so affected by the cleavage or other substrate/biomolecule interaction as to undergo a detectable change. Innumerable examples of detectable assay formats are known from the diagnostic arts which use immunoassay, chromogemc assay, and labeled probe methodologies.
- the optical signal substrate can be a bio- or chemi-luminescent substrate.
- Chemiluminescent substrates for several enzymes are available from Tropix (Bedford, MA).
- the enzymes having known chemiluminescent substrates are alkaline phosphatase, beta-galactosidase, beta-glucouronidase, and beta-glucosidase.
- chromogemc substrates may be used, particularly for certain enzymes such as hydrolytic enzymes.
- the optical signal substrate can be an indolyl derivative, which is enzymatically cleaved f o yield a chromogemc product.
- the optically detectable signal is optical absorbance (including changes in absorbance).
- signal detection can be provided by an absorbance measurement using a spectrophotometer or the like.
- a fluorogenic substrate is used, such that the optically detectable signal is fluorescence.
- Fluorogenic substrates provide high sensitivity for improved detection, as well as alternate detection modes. Hydroxy- and amino- substituted coumarins are the most widely used fluorophores used for preparing fluorogenic substrates.
- a typical coumarin-based fluorogenic substrate is 7- hydroxycoumarin, commonly known as umbelliferone (Umb). Derivatives and analogs of umbelliferone are also used.
- Substrate based on derivative and analogs of fluorescein (such as FDG or C12-FDG) and rhodamine are also used.
- Substrates derived from resorafin e.g., resorufin beta-D -galactopyranoside or resorafin beta-D-glucouronide
- Resorufin-based substrates are useful, for example, in screening for glycosidases, hydrolases and dealkylases.
- Lipophilic derivatives of the foregoing substrates e.g., alkylated derivatives
- Fluorescein and resorafin are available commercially as alkylated derivatives that form products that are relatively insoluble in water (i.e., lipophilic).
- fluorescence imaging can be performed using C12- resorufin galactoside, produced by Molecular Probes (Eugene, OR) as a substrate.
- the particular fluorogenic substrate used may be chosen based on the enzymatic activity being screened.
- the substrates are able to enter the cell and maintain its presence within the cell for a period sufficient for analysis to occur (e.g., once the substrate is in the cell it does not "leak" back out before reacting with the enzyme being screened to an extend sufficient to produce a detectable response). Retention of the substrate in the cell can be enhanced by a variety of techniques.
- the substrate compound is structurally modified by addition of a hydrophobic (e.g., alkyl) tail.
- a solvent such as DMSO or glycerol, can be used to coat the exterior of the cell.
- the substrate can be administered to the cells at reduced temperature, which has been observed to retard leakage of substrates from cells.
- the optical signal substrate can, in some embodiments, be a FRET substrate.
- FRET is a spectroscopic method that can monitor proximity and relative angular orientation of fluorophores.
- a fluorescent indicator system that uses FRET to measure the concentration of a substrate or products includes two fluorescent moieties having emission and excitation spectra that render one a "donor' fluorescent moiety and the other an "acceptor" fluorescent moiety.
- the two fluorescent moieties are chosen such that the excitation spectram of the acceptor fluorescent moiety overlaps with the emission spectrum of the excited moiety (the donor fluorescence moiety).
- the donor moiety is excited by light of appropriate intensity within the excitation spectram of the donor moiety and emits the absorbed energy as fluorescent light.
- the acceptor fluorescent protein moiety is positioned to quench the donor moiety in the excited state, the fluorescence energy is transfened to the acceptor moiety, which can emit a second photon.
- the emission spectra of the donor and acceptor moieties have minimal overlap so that the two emissions can be distinguished. Thus, when acceptor emits fluorescence at longer wavelength that the donor, then the net steady state effect is that the donor's emission is quenched, and the acceptor now emits when excited at the donor's abso ⁇ tion maximum.
- the detectable or optical signal can be measured using, for example, a fluorometer (or the like) to detect fluorescence, including fluorescence polarization, time- resolved fluorescence or FRET.
- excitation radiation from an excitation source having a first wavelength, causes the excitation radiation to excite the sample.
- fluorescence compounds in the sample emit radiation having a wavelength that is different from the excitation wavelength.
- Nolan et al. describes a technique to analyze beta-galactosidase expression in mammalian cells. This technique employs fluorescein- di-beta-D-glactopyranoside (FDG) as a substrate for beta-galactosidase, which releases fluorescein, a product that can be detected by its fluorescence emission upon hydrolysis (Nolan et al., 1991).
- FDG fluorescein- di-beta-D-glactopyranoside
- beta-galactosidase assays may be employed to screen single E. coli cells, expressing recombinant beta-D-galactosidase isolated, for example, from a hyperthermophilic archaeon such as Sulfolobus solfataricus.
- Other reporter genes may be useful as substrates and are known for beta-glucouronidase, alkaline phosphatase, chloramphenical acetyltransferase (CAT) and luciferase.
- the library may, for example, be screened for a specified enzyme activity.
- the enzyme activity screened for may be as a catalyst for the modification of epoxides.
- the recombinant enzymes may then be rescreened for a more specific enzyme activity.
- the library may be screened for a more specialized enzyme activity.
- the library may be screened for a more specialized activity, i.e. the type of bond on which the epoxide hydrolase acts.
- the library may be screened to ascertain those EHs, which act on one or more specified epoxide groups such as mono-substituted epoxides, 2,2-disubstituted epoxides, 2,3-disubstituted epoxides, trisubstituted epoxides and styrene oxides.
- the invention provides a process for activity screening of clones containing selected DNA derived from a microorganism which method includes: [00445] screening a library for a biomolecule of interest or bioactivity of interest, wherein the library includes a plurality of clones, the clones having been prepared by recovering nucleic acids (e.g., genomic DNA) from a mixed population of organisms, enriched populations thereof, or isolates thereof, and transforming a host with the nucleic acids to produce clones which are screened for the biomolecule or bioactivity of interest.
- nucleic acids e.g., genomic DNA
- an enrichment step may be used before activity based screening.
- the enrichment step can be, for example, a biopanning method. This procedure of "biopanning" is described and exemplified in U.S. Patent No. 6,054,002, issued April 25, 2000, which is inco ⁇ orated herein by reference.
- polynucleotides are contained in clones, the clones having been prepared from nucleic acid sequences of a mixed population of organisms, wherein the nucleic acid sequences are used to prepare a gene library of the mixed population of organisms.
- the gene library is screened for a sequence of interest by transfecting a host cell containing the library with at least one nucleic acid sequence having a detectable molecule which is all or a portion of a DNA sequence encoding a bioactivity having a desirable activity and separating the library clones containing the desirable sequence by, for example, a fluorescent based analysis.
- biopanning approach described above can be used to create libraries enriched with clones carrying sequences homologous to a given probe sequence.
- libraries containing clones with inserts of up to 40 kbp can be enriched approximately 1,000 fold after each round of panning. This enables one to reduce the number of clones to be screened after 1 round of biopanning enrichment.
- This approach can be applied to create libraries enriched for clones carrying sequence of interest related to a bioactivity of interest for example polyketide sequences.
- Hybridization screening using high-density filters or biopanning has proven an efficient approach to detect homologues of pathways containing conserved genes.
- Another approach of the present invention is to screen in E. coli for the expression of small molecule ring stractures or "backbones". Because the genes encoding these polycyclic structures can often be expressed in E. coli the small molecule backbone can be manufactured albeit in an inactive form. Bioactivity is conferred upon transferring the molecule or pathway to an appropriate host that expresses the requisite glycosylation and methylation genes that can modify or "decorate" the structure to its active form.
- inactive ring compounds recombinantly expressed in E. coli are detected to identify clones, which are then shuttled to a metabolically rich host, such as Streptomyces, for subsequent production of the bioactive molecule.
- a metabolically rich host such as Streptomyces
- One approach to detect and enrich for clones carrying these structures is to use the capillary screening methods or FACS screening, a procedure described and exemplified in U.S. Ser. No. 08/876,276, filed Jun. 16, 1997.
- Polycyclic ring compounds typically have characteristic fluorescent spectra when excited by ultraviolet light.
- clones expressing these structures can be distinguished from background using a sufficiently sensitive detection method.
- high throughput FACS screening can be utilized to screen for small molecule backbones in E. coli libraries.
- Commercially available FACS machines are capable of screening up to 100,000 clones per second for UV active molecules. These clones can be sorted for further FACS screening or the resident plasmids can be extracted and shuttled to Streptomyces for activity screening.
- organic extracts from candidate clones can be tested for bioactivity by susceptibility screening against test organisms such as Staphylococcus aureus, E. coli, or Saccharomyces cerevisiae.
- FACS screening can be used in this approach by co- encapsulating clones with the test organism.
- An alternative to the above-mentioned screening methods provided by the present invention is an approach termed “mixed extract” screening.
- the “mixed extract” screening approach takes advantage of the fact that the accessory genes needed to confer activity upon the polycyclic backbones are expressed in metabolically rich hosts, such as Streptomyces, and that the enzymes can be extracted and combined with the backbones extracted from E. coli clones to produce the bioactive compound in vitro.
- Enzyme extract preparations from metabolically rich hosts, such as Streptomyces strains, at various growth stages are combined with pools of organic extracts from E. coli libraries and then evaluated for bioactivity.
- Another approach to detect activity in the E. coli clones is to screen for genes that can convert bioactive compounds to different forms.
- Capillary screening for example, can also be used to detect expression of UV fluorescent molecules in metabolically rich hosts, such as Streptomyces.
- Recombinant oxytetracylin retains its diagnostic red fluorescence when produced heterologously in S. lividans TK24.
- Pathway clones which can be identified by the methods and systems of the invention, can thus be screened for polycyclic molecules in a high throughput fashion.
- Recombinant bioactive compounds can also be screened in vivo using "two- hybrid" systems, which can detect enhancers and inhibitors of protein-protein or other interactions such as those between transcription factors and their activators, or receptors and their cognate targets. In this embodiment, both a small molecule pathway and a GFP reporter construct are co-expressed. Clones altered in GFP expression can then be identified and the clone isolated for characterization.
- the present invention also allows for the transfer of cloned pathways derived from uncultivated samples into metabolically rich hosts for heterologous expression and downstream screening for bioactive compounds of interest using a variety of screemng approaches briefly described above.
- DNA can be isolated from positive clones utilizing techniques well known in the art.
- the DNA can then be amplified either in vivo or in vitro by utilizing any of the various amplification techniques known in the art. In vivo amplification would include transformation of the clone(s) or subclone(s) into a viable host, followed by growth of the host. In vitro amplification can be performed using techniques such as the polymerase chain reaction. Once amplified the identified sequences can be "evolved" or sequenced. [00458]
- One advantage afforded by present invention is the ability to manipulate the identified biomolecules or bioactivities to generate and select for encoded variants with altered sequence, activity or specificity.
- Clones found to have biomolecules or bioactivities for which the screen was performed can be subjected to directed mutagenesis to develop new biomolecules or bioactivities with desired properties or to develop modified biomolecules or bioactivities . with particularly desired properties that are absent or less pronounced in nature (e.g., wild-type activity), such as stability to heat or organic solvents.
- Any of the known techniques for directed mutagenesis are applicable to the invention.
- particularly preferred mutagenesis techniques for use in accordance with the invention include those described below.
- a biomolecule e.g., a peptide, protein, or polynucleotide sequence
- a bioactivity e.g., an enzymatic activity
- Such variegation can modify the biomolecule or bioactivity in order to increase or decrease, for example, a polypeptide's activity, specificity, affinity, function, and the like.
- DNA shuffling can be used to increase variegation in a particular sample.
- DNA shuffling is meant to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via non-homologous recombination, such as via cer/lox and/or flp/frt systems and the like (see, for example, U.S. Patent No. 5,939,250, issued to Dr. Jay Short on August 17, 1999, and assigned to Diversa Co ⁇ oration, the disclosure of wliich is inco ⁇ orated herein by reference).
- Various methods for shuffling, mutating or variegating polynucleotide or polypeptide sequences are discussed below.
- Nucleic acid shuffling is a method for in vitro or in vivo homologous recombination of pools of shorter or smaller polynucleotides to produce a polynucleotide or polynucleotides. Mixtures of related nucleic acid sequences or polynucleotides are subjected to sexual PCR to provide random polynucleotides, and reassembled to yield a library or mixed population of recombinant hybrid nucleic acid molecules or polynucleotides. In contrast to cassette mutagenesis, only shuffling and error-prone PCR allow one to mutate a pool of sequences blindly (without sequence information other than primers).
- the advantage of the mutagenic shuffling of the invention over error-prone PCR alone for repeated selection can best be explained as follows.
- the initial library of selected or pooled sequences can consist of related sequences of diverse origin or can be derived by any type of mutagenesis (including shuffling) of a single gene.
- a collection of selected sequences is obtained after the first round of activity selection.
- Shuffling allows the free combinatorial association of all of the related sequences, for example.
- This method differs from error-prone PCR, in that it is an inverse chain reaction. In error-prone PCR, the number of polymerase start sites and the number of molecules grows exponentially.
- the sequence of the polymerase start sites and the sequence of the molecules remains essentially the same.
- the number of start sites and the number (but not size) of the random polynucleotides decreases over time.
- the theoretical endpoint is a single, large concatemeric molecule.
- a pool of 100 different polypeptide sequences can be permutated in up to 103 different ways. This large number of permutations cannot be represented in a single library of DNA sequences. Accordingly, it is contemplated that multiple cycles of DNA shuffling and selection may be required depending on the length of the sequence and the sequence diversity desired. Error-prone PCR, in contrast, keeps all the selected sequences in the same relative orientation, generating a much smaller mutant cloud.
- the template polynucleotide which may be used in the methods of the invention may be DNA or RNA. It may be of various lengths depending on the size of the gene or shorter or smaller polynucleotide to be recombined or reassembled.
- the template polynucleotide is from 50 bp to 50 kb. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest can be used in the methods of the invention, and in fact have been successfully used.
- the template polynucleotide may be obtained by amplification using the PCR reaction (US Patent Nos. 4,683,202 and 4,683,195) or other amplification or cloning methods. However, the removal of free primers from the PCR products before subjecting them to pooling of the PCR products and sexual PCR may provide more efficient results. Failure to adequately remove the primers from the original pool before sexual PCR can lead to a low frequency of crossover clones.
- the template polynucleotide often is double-stranded.
- a double-stranded nucleic acid molecule is recommended to ensure that regions of the resulting single- stranded polynucleotides are complementary to each other and thus can hybridize to form a double-stranded molecule.
- single-stranded or double-stranded nucleic acid polynucleotides having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide, at this step. It is also contemplated that two different but related polynucleotide templates can be mixed at this step.
- the double-stranded polynucleotide template and any added double- or single-stranded polynucleotides are subjected to sexual PCR which includes slowing or halting to provide a mixture of from about 5 bp to 5 kb or more.
- the size of the random polynucleotides is from about 10 bp to 1000 bp, more preferably the size of the polynucleotides is from about 20 bp to 500 bp.
- double-stranded nucleic acid having multiple nicks may be used in the methods of the invention.
- a nick is a break in one strand of the double-stranded nucleic acid.
- the distance between such nicks is preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp. This can provide areas of self- priming to produce shorter or smaller polynucleotides to be included with the polynucleotides resulting from random primers, for example.
- the concentration of any one specific polynucleotide will not be greater than 1% by weight of the total polynucleotides, more preferably the concentration of any one specific nucleic acid sequence will not be greater than 0.1% by weight of the total nucleic acid.
- the number of different specific polynucleotides in the mixture will be at least about 100, preferably at least about 500, and more preferably at least about 1000.
- single-stranded or double-stranded polynucleotides, either synthetic or natural, may be added to the random double-stranded shorter or smaller polynucleotides in order to increase the heterogeneity of the mixture of polynucleotides.
- populations of double-stranded randomly broken polynucleotides may be mixed or combined at this step with the polynucleotides from the sexual PCR process and optionally subjected to one or more additional sexual PCR cycles.
- single-stranded or double-stranded polynucleotides having a region of identity to the template polynucleotide and a region of heterology to the template polynucleotide may be added in a 20 fold excess by weight as compared to the total nucleic acid, more preferably the single-stranded polynucleotides may be added in a 10 fold excess by weight as compared to the total nucleic acid.
- populations of polynucleotides from each of the templates may be combined at a ratio of less than about 1:100, more preferably the ratio is less than about 1 :40.
- a backcross of the wild-type polynucleotide with a population of mutated polynucleotide may be desired to eliminate neutral mutations (e.g., mutations yielding an insubstantial alteration in the phenotypic property being selected for).
- the ratio of randomly provided wild-type polynucleotides which may be added to the randomly provided sexual PCR cycle hybrid polynucleotides is approximately 1:1 to about 100:1, and more preferably from 1 : 1 to 40: 1.
- the mixed population of random polynucleotides are denatured to form single-stranded polynucleotides and then re-annealed. Only those single-stranded polynucleotides having regions of homology with other single-stranded polynucleotides will re-anneal.
- the random polynucleotides may be denatured by heating.
- One skilled in the art could determine the conditions necessary to completely denature the double-stranded nucleic acid.
- the temperature is from 80°C to 100°C, more preferably the temperature is from 90°C to 96°C.
- Other methods, which may be used to denature the polynucleotides include pressure and pH.
- the polynucleotides may be re-annealed by cooling.
- the temperature is from 20°C to 75°C, more preferably the temperature is from 40°C to 65°C. If a high frequency of crossovers is needed based on an average of only 4 consecutive bases of homology, recombination can be forced by using a low annealing temperature, although the process becomes more difficult.
- the degree of renaturation, which occurs will depend on the degree of homology between the population of single-stranded polynucleotides.
- Renaturation can be accelerated by the addition of polyethylene glycol ("PEG”) or salt.
- PEG polyethylene glycol
- the salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mm.
- the salt may be KCl or NaCl.
- the concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.
- the annealed polynucleotides are next incubated in the presence of a nucleic acid polymerase and dNTP's (i.e. dATP, dCTP, DGTP and dTTP).
- a nucleic acid polymerase i.e. dATP, dCTP, DGTP and dTTP.
- the nucleic acid polymerase may be the Klenow fragment, the Taq polymerase or any other DNA polymerase known in the art.
- the approach to be used for the assembly depends on the minimum degree of homology that should still yield crossovers. If the areas of identity are large, Taq polymerase can be used with an annealing temperature of between 45D65 DC. If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20° " 30°C. One skilled in the art could vary the temperature of annealing to increase the number of crossovers achieved. [00485] The polymerase may be added to the random polynucleotides prior to annealing, simultaneously with annealing or after annealing.
- the cycle of denaturation, renaturation and incubation in the presence of polymerase is referred to herein as shuffling or reassembly of the nucleic acid.
- This cycle is repeated for a desired number of times.
- the cycle is repeated from 2 to 50 times, more preferably the sequence is repeated from 10 to 40 times.
- the resulting nucleic acid is a larger double-stranded polynucleotide of from about 50 bp to about 100 kb, preferably the larger polynucleotide is from 500 bp to 50 kb.
- These larger polynucleotides may contain a number of copies of a polynucleotide having the same size as the template polynucleotide in tandem. This concatemeric polynucleotide is then denatured into single copies of the template polynucleotide. The result will be a population of polynucleotides of approximately the same size as the template polynucleotide. The population will be a mixed population where single or double-stranded polynucleotides having an area of identity and an area of heterology have been added to the template polynucleotide prior to shuffling. These polynucleotides are then cloned into the appropriate vector and the ligation mixture used to transform bacteria.
- the single polynucleotides may be obtained from the larger concatemeric polynucleotide by amplification of the single polynucleotide prior to cloning by a variety of methods including PCR (US Patent Nos. 4,683,195 and 4,683,202), rather than by digestion of the concatemer.
- the vector used for cloning is not critical provided that it will accept a polynucleotide of the desired size. If expression of the particular polynucleotide is desired, the cloning vehicle should further comprise transcription and translation signals next to the site of insertion of the polynucleotide to allow expression of the polynucleotide in the host cell.
- the resulting bacterial population will include a number of recombinant polynucleotides having random mutations. This mixed population may be tested to identify the desired recombinant polynucleotides. The method of selection will depend on the polynucleotide desired.
- a polynucleotide identified by the methods of described herein, encodes a protein with a first binding affinity
- subsequent mutated (e.g., shuffled) sequences having an increased binding efficiency to a ligand may be desired.
- the proteins expressed by each of the portions of the polynucleotides in the population or library may be tested for their ability to bind to the ligand by methods known in the art (i.e. panning, affinity chromatography).
- the proteins expressed by each of the polynucleotides in the population or library may be tested for their ability to confer drag resistance to the host organism.
- One skilled in the art given knowledge of the desired protein, could readily test the population to identify polynucleotides, which confer the desired properties onto the protein.
- a phage display system in which fragments of the protein are expressed as fusion proteins on the phage surface (Pharmacia, Milwaukee WI).
- the recombinant DNA molecules are cloned into the phage DNA at a site, which results in the transcription of a fusion protein a portion of which is encoded by the recombinant DNA molecule.
- the phage containing the recombinant nucleic acid molecule undergoes replication and transcription in the cell.
- the leader sequence of the fusion protein directs the transport of the fusion protein to the tip of the phage particle.
- the fusion protein which is partially encoded by the recombinant DNA molecule is displayed on the phage particle for detection and selection by the methods described above.
- nucleic acid shuffling may be conducted with polynucleotides from a sub-population of the first population, which sub-population contains DNA encoding the desired recombinant protein. In this manner, proteins with even higher binding affinities or enzymatic activity could be achieved.
- nucleic acid shuffling may be conducted with a mixture of wild type polynucleotides and a sub-population of nucleic acid from the first or subsequent rounds of nucleic acid shuffling in order to remove any silent mutations from the sub-population.
- any source of nucleic acid, in a purified form can be utilized as the starting nucleic acid.
- the process may employ DNA or RNA including messenger RNA, which DNA or RNA may be single or double stranded.
- a DNA D RNA hybrid which contains one strand of each may be utilized.
- the nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated.
- the specific nucleic acid sequence is from 50 to 50,000 base pairs. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest may be used in the methods of the invention.
- Any specific nucleic acid sequence can be used to produce the population of hybrids by the present process. It is only necessary that a small population of hybrid sequences of the specific nucleic acid sequence exist or be available for the present process.
- a population of specific nucleic acid sequences having mutations may be created by a number of different methods. Mutations may be created by error-prone PCR. Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Alternatively, mutations can be introduced into the template polynucleotide by oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the polynucleotide using restriction enzyme digestion and is replaced with a synthetic polynucleotide in which various bases have been altered from the original sequence.
- the polynucleotide sequence can also be altered by chemical mutagenesis.
- Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
- Other agents which are analogues of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these agents are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence.
- Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.
- Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light.
- plasmid polynucleotides so mutagenized are introduced into E. coli and propagated as a pool or library of hybrid plasmids.
- a small mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species (i.e., cognate genes). Alternatively, they may be related DNA sequences found within one species, for example, the immunoglobulin genes.
- the polynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well known in the art.
- the choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of the invention.
- the templates of the invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzaviras, he ⁇ esviruses, reoviruses, paramyxovirases, and the like), or selected portions thereof (e.g., coat protem, spike glycoprotein, capsid protein).
- cosmids and phagemids are preferred where the specific nucleic acid sequence to be mutated is larger because these vectors are able to stably propagate large polynucleotides.
- a mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified. Utility can be readily determined by screening expressed polypeptides.
- the DNA shuffling method of the invention can be performed blindly on a pool of unknown sequences.
- any sequence mixture can be inco ⁇ orated at any specific position into another sequence mixture.
- mixtures of synthetic oligonucleotides, PCR polynucleotides or even whole genes can be mixed into another sequence library at defined positions.
- the insertion of one sequence (mixture) is independent from the insertion of a sequence in another part of the template.
- the degree of recombination, the homology required, and the diversity of the library can be independently and simultaneously varied along the length of the reassembled DNA.
- Shuffling requires the presence of homologous regions separating regions of diversity. Scaffold-like protein structures may be particularly suitable for shuffling.
- the conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding. Examples of such scaffolds are the immunoglobulin beta-barrel, and the four helix bundle which are well- known in the art. This shuffling can be used to create scaffold like proteins with various combinations of mutated sequences for binding.
- the equivalents of some standard genetic matings may also be performed by shuffling in vitro.
- a "molecular backcross" can be performed by repeatedly mixing the hybrid's nucleic acid with the wild type nucleic acid while selecting for the mutations of interest.
- this approach can be used to combine phenotypes from different sources into a background of choice. It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (e.g., irnmunogenicity).
- unselected characteristics e.g., irnmunogenicity
- a 100 fold amplification in the copy number and an average polynucleotide size of greater than 50 kb may be obtained when only random polynucleotides are used. It is thought that the larger concatamer is generated by overlap of many smaller polynucleotides. The quality of specific PCR products obtained using synthetic primers will be indistinguishable from the product obtained from unamplified DNA. It is expected that this approach will be useful for the mapping of genomes.
- the polynucleotide to be shuffled can be produced as random or non-random polynucleotides, at the discretion of the practitioner.
- the invention provides a method of shuffling that is applicable to a wide range of polynucleotide sizes and types, including the step of generating polynucleotide monomers to be used as building blocks in the reassembly of a larger polynucleotide.
- the building blocks can be fragments of genes or they can be comprised of entire genes or gene pathways, or any combination thereof.
- a mixed population of a specific nucleic acid sequence is introduced into bacterial or eukaryotic cells under conditions such that at least two different nucleic acid sequences are present in each host cell.
- the polynucleotides can be introduced into the host cells by a variety of different methods.
- the host cells can be transformed with the smaller polynucleotides using methods known in the art, for example treatment with calcium chloride. If the polynucleotides are inserted into a phage genome, the host cell can be transfected with the recombinant phage genome having the specific nucleic acid sequences.
- the nucleic acid sequences can be introduced into the host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.
- nucleic acid sequences will be present in vectors, which are capable of stably replicating the sequence in the host cell.
- the vectors will encode a marker gene such that host cells having the vector can be selected. This ensures that the mutated specific nucleic acid sequence can be recovered after introduction into the host cell.
- the entire mixed population of the specific nucleic acid sequences need not be present on a vector sequence. Rather only a sufficient number of sequences need be cloned into vectors to ensure that after introduction of the polynucleotides into the host cells each host cell contains one vector having at least one specific nucleic acid sequence present therein. It is also contemplated that rather than having a subset of the population of the specific nucleic acids sequences cloned into vectors, this subset may be already stably integrated into the host cell.
- the host cell transformants are placed under selection to identify those host cell transformants, which contain mutated specific nucleic acid sequences having the qualities desired. For example, if increased resistance to a particular drug is desired then the transformed host cells may be subjected to increased concentrations of the particular drag and those transformants producing mutated proteins able to confer increased drug resistance will be selected. If the enhanced ability of a particular protein to bind to a receptor is desired, then expression of the protein can be induced from the transformants and the resulting protein assayed in a ligand binding assay by methods known in the art to identify that subset of the mutated population which shows enhanced binding to the ligand. Alternatively, the protein can be expressed in another system to ensure proper processing.
- the recombined specific nucleic acid sequences may be mixed with the original mutated specific nucleic acid sequences (parent sequences) and the cycle repeated as described above.
- This cycle can be repeated a number of times as desired.
- a molecular backcross can be performed by mixing the desired specific nucleic acid sequences with a large number of the wild type sequences, such that at least one wild type nucleic acid sequence and a mutated nucleic acid sequence are present in the same host cell after transformation. Recombination with the wild type specific nucleic acid sequence will eliminate those neutral mutations that may affect unselected characteristics such as immunogenicity but not the selected characteristics.
- a subset of specific nucleic acid sequences can be generated as smaller polynucleotides by slowing or halting their PCR amplification prior to introduction into the host cell.
- the size of the polynucleotides must be large enough to contain some regions of identity with the other sequences so as to homologously recombine with the other sequences.
- the size of the polynucleotides will range from 0.03 kb to 100 kb more preferably from 0.2 kb to 10 kb. It is also contemplated that in subsequent rounds, all of the specific nucleic acid sequences other than the sequences selected from the previous round may be utilized to generate PCR polynucleotides prior to introduction into the host cells.
- the shorter polynucleotide sequences can be single-stranded or double- stranded.
- the reaction conditions suitable for separating the strands of nucleic acid are well known in the art.
- the initial pool or population of mutated template nucleic acid is cloned into a vector capable of replicating in a bacteria such as E. coli.
- the particular vector is not essential, so long as it is capable of autonomous replication in E. coli.
- the vector is designed to allow the expression and production of any protein encoded by the mutated specific nucleic acid linked to the vector. It is also preferred that the vector contain a gene encoding for a selectable marker.
- the population of vectors containing the pool of mutated nucleic acid sequences is introduced into the ⁇ . coli host cells.
- the vector nucleic acid sequences may be introduced by transformation, transfection or infection in the case of phage.
- the concentration of vectors used to transform the bacteria is such that a number of vectors is introduced into each cell. Once present in the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors. This results in the generation of hybrids (daughters) having a combination of mutations, which differ from the original parent mutated sequences.
- the host cells are then clonally replicated and selected for the marker gene present on the vector. Only those cells having a plasmid will grow under the selection. The host cells, which contain a vector are then tested for the presence of favorable mutations.
- nucleic acid is isolated either aheady linked to the vector or separated from the vector. This nucleic acid is then mixed with the first or parent population of nucleic acids and the cycle is repeated.
- the parent mutated specific nucleic acid population is introduced into the host cells already containing the daughter nucleic acids. Recombination is allowed to occur in the cells and the next generation of recombinants, or granddaughters are selected by the methods described above. This cycle can be repeated a number of times until the nucleic acid or peptide having the desired characteristics is obtained. It is contemplated that in subsequent cycles, the population of mutated sequences, which are added to the hybrids may come from the parental hybrids or any subsequent generation.
- the invention provides a method of conducting a "molecular" backcross of the obtained recombinant specific nucleic acid in order to eliminate any neutral mutations.
- Neutral mutations are those mutations, which do not confer onto the nucleic acid or peptide the desired properties. Such mutations may however confer on the nucleic acid or peptide undesirable characteristics. Accordingly, it is desirable to eliminate such neutral mutations.
- the method of the invention provides a means of doing so.
- the nucleic acid, having the desired characteristics is obtained by the methods of the embodiments, the nucleic acid, the vector having the nucleic acid or the host cell containing the vector and nucleic acid is isolated. [00526] The nucleic acid or vector is then introduced into the host cell with a large excess of the wild type nucleic acid. The nucleic acid of the hybrid and the nucleic acid of the wild type sequence are allowed to recombine. The resulting recombinants are placed under the same selection as the hybrid nucleic acid. Only those recombinants, which retained the desired characteristics will be selected. Any silent mutations which do not provide the desired characteristics will be lost through recombination with the wild type DNA.
- the invention provides for a method for shuffling, assembling, reassembling, recombining, and/or concatenating at least two polynucleotides to form a progeny polynucleotide (e.g., a chimeric progeny polynucleotide that can be expressed to produce a polypeptide or a gene pathway).
- a progeny polynucleotide e.g., a chimeric progeny polynucleotide that can be expressed to produce a polypeptide or a gene pathway.
- a double stranded polynucleotide (e.g., two single stranded sequences hybridized to each other as hybridization partners) is treated with an exonuclease to liberate nucleotides from one of the two strands, leaving the remaining strand free of its original partner so that, if desired, the remaining strand may be used to achieve hybridization to another partner.
- a double stranded polynucleotide end (that may be part of- or connected to - a polynucleotide or a non-polynucleotide sequence) is subjected to a source of exonuclease activity.
- Enzyme with 3' exonuclease activity, an enzyme with 5' exonuclease activity, an enzyme with both 3' exonuclease activity and 5' exonuclease activity, and any combination thereof can be used in the invention.
- An exonuclease can be used to liberate nucleotides from one or both ends of a linear double stranded polynucleotide, and from one to all ends of a branched polynucleotide having more than two ends.
- a non-enzymatic step may be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks that is comprised of subjecting a working sample to denaturing (or "melting") conditions (for example, by changing temperature, pH, and /or salinity conditions) so as to melt a working set of double stranded polynucleotides into single polynucleotide strands.
- denaturing or "melting”
- the single polynucleotide strands participate to some extent in annealment with different hybridization partners (i.e.
- the invention further provides an exonuclease-based approach requiring no denaturation - rather, the avoidance of denaturing conditions and the maintenance of double stranded polynucleotide substrates in annealed (i.e. non-denatured) state are necessary conditions for the action of exonucleases (e.g., exonuclease IE and red alpha gene product).
- exonucleases e.g., exonuclease IE and red alpha gene product.
- the generation of single stranded polynucleotide sequences capable of hybridizing to other single stranded polynucleotide sequences is the result of covalent cleavage - and hence sequence destruction - in one of the hybridization partners.
- an exonuclease III enzyme may be used to enzymatically liberate 3' terminal nucleotides in one hybridization strand (to achieve covalent hydrolysis in that polynucleotide strand); and this favors hybridization of the remaining single strand to a new partner (since its former partner was subjected to covalent cleavage).
- enzymes can be discovered, optimized (e.g., engineered by directed evolution), or both discovered and optimized specifically for the instantly disclosed approach that have more optimal rates and/or more highly specific activities &/or greater lack of unwanted activities. In fact it is expected that the invention may encourage the discovery and/or development of such designer enzymes.
- exonuclease III may be rendered susceptible to the exonuclease action of exonuclease III by a variety of means; for example, it may be blunted by treatment with a polymerase, cleaved to provide a blunt end or a 5' overhang, joined (ligated or hybridized) to another double stranded polynucleotide to provide a blunt end or a 5' overhang, hybridized to a single stranded polynucleotide to provide a blunt end or a 5' overhang, or modified by any of a variety of means).
- an exonuclease may be allowed to act on one or on both ends of a linear double stranded polynucleotide and proceed to completion, to near completion, or to partial completion.
- the exonuclease action is allowed to go to completion, the result will be that the length of each 5' overhang will be extend far towards the middle region of the polynucleotide in the direction of what might be considered a "rendezvous point" (which may be somewhere near the polynucleotide midpoint).
- the exonuclease-mediated approach is useful for shuffling, assembling and/or reassembling, recombining, and concatenating polynucleotide building blocks.
- the polynucleotide building blocks can be up to ten bases long or tens of bases long or hundreds of bases long or thousands of bases long or tens of thousands of bases long or hundreds of thousands of bases long or millions of bases long or even longer.
- Substrates for an exonuclease may be generated by subjecting a double stranded polynucleotide to fragmentation.
- Fragmentation may be achieved by mechanical means (e.g., shearing, sonication, and the like), by enzymatic means (e.g., using restriction enzymes), and by any combination thereof. Fragments of a larger polynucleotide may also be generated by polymerase-mediated synthesis.
- Additional examples of enzymes with exonuclease activity include red-alpha and venom phosphodiesterases. Red alpha (red alpha gene product (also referred to as lambda exonuclease) is of bacteriophage alpha origin.
- Red alpha gene product acts processively from 5 '-phosphorylated termini to liberate mononucleotides from duplex DNA (Takahashi & Kobayashi, 1990). Venom phosphodiesterases (Laskowski, 1980) is capable of rapidly opening supercoiled DNA.
- the design of nucleic acid building blocks is obtained upon analysis of the sequences of a set of progenitor nucleic acid templates that serve as a basis for producing a progeny set of finalized chimeric nucleic acid molecules.
- progenitor nucleic acid templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, i.e. chimerized or shuffled.
- the invention provides for the chimerization of a family of related genes and their encoded family of related products.
- the encoded products are enzymes.
- the sequences of a plurality of progenitor nucleic acid templates identified using the methods of the invention are aligned in order to select one or more demarcation points, which demarcation points can be located at an area of homology.
- the demarcation points can be used to delineate the boundaries of nucleic acid building blocks to be generated.
- the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the progeny molecules.
- a demarcation point is an area of homology (comprised of at least one homologous nucleotide base) shared by at least two progenitor templates, but the demarcation point can be an area of homology that is shared by at least half of the progenitor templates, at least two thirds of the progenitor templates, at least three fourths of the progenitor templates, and preferably at almost all of the progenitor templates. Even more preferably still a demarcation point is an area of homology that is shared by all of the progenitor templates.
- the ligation reassembly process is performed exhaustively in order to generate an exhaustive library.
- all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.
- the assembly order i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid
- the assembly order is by design (or non-stochastic). Because of the non- stochastic nature of the invention, the possibility of unwanted side products is greatly reduced.
- the invention provides that, the ligation reassembly process is performed systematically, for example in order to generate a systematically compartmentalized library, with compartments that can be screened systematically, e.g., one by one.
- the invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, an experimental design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, it allows a potentially very large number of progeny molecules to be examined systematically in smaller groups.
- the instant invention provides for the generation of a library (or set) comprised of a large number of progeny molecules.
- the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.
- such a generated library is comprised of greater than 10 to greater than 10 1000 different progeny molecular species.
- a set of finalized chimeric nucleic acid molecules, produced as described is comprised of a polynucleotide encoding a polypeptide.
- this polynucleotide is a gene, which may be a man-made gene.
- this polynucleotide is a gene pathway, which may be a man- made gene pathway.
- the invention provides that one or more man-made genes generated by the invention may be inco ⁇ orated into a man-made gene pathway, such as pathway operable in a eukaryotic organism (including a plant).
- the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g., by mutagenesis) or in an in vivo process (e.g., by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a demarcation point.
- nucleotides e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences
- the invention provides that a nucleic acid building block can be used to introduce an intron.
- the invention provides that functional introns may be introduced into a man-made gene of the invention.
- the invention also provides that functional introns may be introduced into a man-made gene pathway of the invention.
- the invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced intron(s).
- the invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced intron(s).
- the artificially introduced intron(s) are functional in one or more host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.
- the invention provides a process of producing man- made intron-containing polynucleotides to be introduced into host organisms for recombination and/or splicing.
- a man-made gene produced using the invention can also serve as a substrate for recombination with another nucleic acid.
- a man-made gene pathway produced using the invention can also serve as a substrate for recombination with another nucleic acid.
- the recombination is facilitated by, or occurs at, areas of homology between the man-made intron-containing gene and a nucleic acid with serves as a recombination partner.
- the recombination partner may also be a nucleic acid generated by the invention, including a man-made gene or a man-made gene pathway. Recombination may be facilitated by or may occur at areas of homology that exist at the one (or more) artificially introduced intron(s) in the man-made gene.
- the synthetic ligation reassembly method of the invention utilizes a plurality of nucleic acid building blocks, each of which preferably has two ligatable ends.
- the two ligatable ends on each nucleic acid building block may be two blunt ends (i.e. each having an overhang of zero nucleotides), or preferably one blunt end and one overhang, or more preferably still two overhangs.
- An overhang for this pu ⁇ ose may be a 3 ' overhang or a 5' overhang.
- a nucleic acid building block may have a 3' overhang or alternatively a 5' overhang or alternatively two 3' overhangs or alternatively two 5' overhangs.
- the overall order in which the nucleic acid building blocks are assembled to form a finalized chimeric nucleic acid molecule is determined by pu ⁇ oseful experimental design and is not random.
- a nucleic acid building block is generated by chemical synthesis of two single-stranded nucleic acids (also referred to as single-stranded oligos) and contacting them so as to allow them to anneal to form a double-stranded nucleic acid building block.
- a double-stranded nucleic acid building block can be of variable size.
- the sizes of these building blocks can be small or large. Preferred sizes for building block range from 1 base pair (not including any overhangs) to 100,000 base pairs (not including any overhangs). Other preferred size ranges are also provided, which have lower limits of from 1 bp to 10,000 bp (including every integer value in between), and upper limits of from 2 bp to 100, 000 bp (including every integer value in between).
- a double-stranded nucleic acid building block is generated by first generating two single stranded nucleic acids and allowing them to anneal to form a double-stranded nucleic acid building block.
- the two strands of a double-stranded nucleic acid building block may be complementary at every nucleotide apart from any that form an overhang; thus containing no mismatches, apart from any overhang(s).
- the two strands of a double-stranded nucleic acid building block are complementary at fewer than every nucleotide apart from any that form an overhang.
- a double-stranded nucleic acid building block can be used to introduce codon degeneracy.
- the codon degeneracy is introduced using the site-saturation mutagenesis described herein, using one or more N,N,G/T cassettes or alternatively using one or more N,N,N cassettes.
- the in vivo recombination method of the invention can be performed blindly on a pool of unknown hybrids or alleles of a specific polynucleotide or sequence. However, it is not necessary to know the actual DNA or RNA sequence of the specific polynucleotide.
- the approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin I, antibodies, tPA and growth hormone.
- This approach may be used to generate proteins having altered specificity or activity.
- the approach may also be useful for the generation of hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions or 51 untranslated regions of genes.
- This approach may be used to generate genes having increased rates of expression.
- This approach may also be useful in the study of repetitive DNA sequences.
- this approach maybe useful to mutate ribozymes or aptamers.
- the invention provides a method for selecting a subset of polynucleotides from a starting set of polynucleotides, which method is based on the ability to discriminate one or more selectable features (or selection markers) present anywhere in a working polynucleotide, so as to allow one to perform selection for (positive selection) and or against (negative selection) each selectable polynucleotide.
- a method is provided termed end-selection, which method is based on the use of a selection marker located in part or entirely in a terminal region of a selectable polynucleotide, and such a selection marker may be termed an "end-selection marker".
- End-selection may be based on detection of naturally occurring sequences or on detection of sequences introduced experimentally (including by any mutagenesis procedure mentioned herein and not mentioned herein) or on both, even within the same polynucleotide.
- An end-selection marker can be a structural selection marker or a functional selection marker or both a structural and a functional selection marker.
- An end-selection marker may be comprised of a polynucleotide sequence or of a polypeptide sequence or of any chemical structure or of any biological or biochemical tag, including markers that can be selected using methods based on the detection of radioactivity, of enzymatic activity, of fluorescence, of any optical feature, of a magnetic property (e.g., using magnetic beads), of immunoreactivity, and of hybridization.
- End-selection may be applied in combination with any method for performing mutagenesis. Such mutagenesis methods include, but are not limited to, methods described herein (supra and infra).
- Such methods include, by way of non-limiting exemplification, any method that may be referred herein or by others in the art by any of the following terms: “saturation mutagenesis”, “shuffling”, “recombination”, “reassembly”, “error-prone PCR”, “assembly PCR”, “sexual PCR”, “crossover PCR”, “oligonucleotide primer-directed mutagenesis”, “recursive (and/or exponential) ensemble mutagenesis (see Arkin and Youvan, 1992)”, “cassette mutagenesis”, “in vivo mutagenesis”, and “in vitro mutagenesis”.
- end-selection may be performed on molecules produced by any mutagenesis and/or amplification method (see, e.g., Arnold, 1993; Caldwell and Joyce, 1992; Stemmer, 1994) following which method it is desirable to select for (including to screen for the presence of) desirable progeny molecules.
- end-selection may be applied to a polynucleotide apart from any mutagenesis method.
- end-selection as provided herein, can be used in order to facilitate a cloning step, such as a step of ligation to another polynucleotide (including ligation to a vector).
- the invention thus provides for end- selection as a means to facilitate library construction, selection and/or enrichment for desirable polynucleotides, and cloning in general.
- end-selection can be based on (positive) selection for a polynucleotide; alternatively end-selection can be based on (negative) selection against a polynucleotide; and alternatively still, end-selection can be based on both (positive) selection for, and on (negative) selection against, a polynucleotide.
- End-selection, along with other methods of selection and/or screening, can be performed in an iterative fashion, with any combination of like or unlike selection and/or screening methods and mutagenesis or directed evolution methods, all of which can be performed in an iterative fashion and in any order, combination, and permutation.
- end- selection may also be used to select a polynucleotide in a: circular (e.g., a plasmid or any other circular vector or any other polynucleotide that is partly circular), and/or branched, and/or modified or substituted with any chemical group or moiety.
- end-selection of a linear polynucleotide is performed using a general approach based on the presence of at least one end-selection marker located at or near a polynucleotide end or terminus (that can be either a 5' end or a 3' end).
- end-selection is based on selection for a specific sequence at or near a terminus such as, but not limited to, a sequence recognized by an enzyme that recognizes a polynucleotide sequence.
- An enzyme that recognizes and catalyzes a chemical modification of a polynucleotide is referred to herein as a polynucleotide-acting enzyme.
- polynucleotide-acting enzymes are exemplified non-exclusively by enzymes with polynucleotide-cleaving activity, enzymes with polynucleotide-methylating activity, enzymes with polynucleotide-ligating activity, and enzymes with a plurality of distinguishable enzymatic activities (including non-exclusively, e.g., both polynucleotide-cleaving activity and polynucleotide-ligating activity).
- relevant polynucleotide-acting enzymes include any enzymes identifiable by one skilled in the art (e.g., commercially available) or that may be developed in the future, though currently unavailable, that are useful for generating a ligation compatible end, preferably a sticky end, in a polynucleotide. It may be preferable to use restriction sites that are not contained, or alternatively that are not expected to be contained, or alternatively that are unlikely to be contained (e.g., when sequence information regarding a working polynucleotide is incomplete) internally in a polynucleotide to be subjected to end-selection.
- a partial digestion reaction i.e. a digestion reaction that proceeds to partial completion
- a digestion reaction can be used to achieve digestion at a recognition site in a terminal region while sparing a susceptible restriction site that occurs internally in a polynucleotide and that is recognized by the same enzyme.
- partial digest are useful because it is appreciated that certain enzymes show preferential cleavage of the same recognition sequence depending on the location and environment in which the recognition sequence occurs.
- a useful end-selection marker is a terminal sequence that is recognized by a polynucleotide-acting enzyme that recognizes a specific polynucleotide sequence.
- useful polynucleotide-acting enzymes also include other enzymes in addition to classic type II restriction enzymes.
- useful polynucleotide-acting enzymes also include gyrases (e.g., topoisomerases), helicases, recombinases, relaxases, and any enzymes related thereto.
- end-selection can be used to distinguish and separate parental template molecules (e.g., to be subjected to mutagenesis) from progeny molecules (e.g., generated by mutagenesis).
- a first set of primers lacking in a topoisomerase I recognition site, can be used to modify the terminal regions of the parental molecules (e.g., in polymerase-based amplification).
- a different second set of primers (e.g., having a topoisomerase I recognition site) can then be used to generate mutated progeny molecules (e.g., using any polynucleotide chimerization method, such as interrupted synthesis, template-switching polymerase-based amplification, or interrupted synthesis; or using saturation mutagenesis; or using any other method for introducing a topoisomerase I recognition site into a mutagenized progeny molecule) from the amplified template molecules.
- the use of topoisomerase I-based end-selection can then facilitate, not only discernment, but selective topoisomerase I-based ligation of the desired progeny molecules.
- the present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by peptide display methods, wherein an associated polynucleotide encodes a displayed peptide which is screened for a phenotype (e.g., for affinity for a predetermined receptor (ligand).
- a phenotype e.g., for affinity for a predetermined receptor (ligand).
- peptide stractures including the primary amino acid sequences, of peptides or peptidomimetics that interact with biological macromolecules.
- One method of identifying peptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor) involves the screening of a large library or peptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the peptide.
- a predetermined biological macromolecule e.g., a receptor
- a well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein.
- the bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule (e.g., a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule.
- the bacteriophage particles i.e., library members
- which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage sub-population for a subsequent round of affinity enrichment and phage replication.
- the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined macromolecule (e.g., receptor).
- the predetermined macromolecule e.g., receptor
- the present invention also provides random, pseudorandom, and defined sequence framework peptide libraries and methods for generating and screening those libraries to identify useful compounds (e.g., peptides, including single-chain antibodies) that bind to receptor molecules or epitopes of interest or gene products that modify peptides or RNA in a desired fashion.
- useful compounds e.g., peptides, including single-chain antibodies
- the random, pseudorandom, and defined sequence framework peptides are produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized.
- the mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsulation in a phage particle or inco ⁇ oration in a cell.
- a significant advantage of the present invention is that no prior information regarding an expected ligand structure is required to isolate peptide ligands or antibodies of interest.
- the peptide identified can have biological activity, which is meant to include at least specific binding affinity for a selected receptor molecule and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like.
- the invention also provides a method for shuffling a pool of polynucleotide sequences identified by the methods of the invention and selected by affinity screening a library of polysomes displaying nascent peptides (including single-chain antibodies) for library members which bind to a predetermined receptor (e.g., a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as hetero-dimers and the like) or epitope (e.g., an immobilized protein, glycoprotein, oligosaccharide, and the like).
- a predetermined receptor e.g., a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as hetero-dimers and the like
- Polynucleotide sequences selected in a first selection round are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.
- the recombined selected polynucleotide sequences are subjected to at least one subsequent selection round.
- the polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and or subjected to one or more additional rounds of shuffling and subsequent selection.
- Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral sequences (i.e., having insubstantial functional effect on binding), such as for example by back-crossing with a wild-type or naturally-occurring sequence substantially identical to a selected sequence to produce native-like functional peptides, which may be less imrnunogenic. Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined receptor (ligand).
- the sequences Prior to or concomitant with the shuffling of selected sequences, the sequences can be mutagenized.
- selected library members are cloned in a prokaryotic vector (e.g., plasmid, phagemid, or bacteriophage) wherein a collection of individual colonies (or plaques) representing discrete library members is produced.
- Individual selected library members can then be manipulated (e.g., by site-directed mutagenesis, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like) to generate a collection of library members representing a kemal of sequence diversity based on the sequence of the selected library member.
- sequence of an individual selected library member or pool can be manipulated to inco ⁇ orate random mutation, pseudorandom mutation, defined kemal mutation (i.e., comprising variant and invariant residue positions and/or comprising variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), codon-based mutation, and the like, either segmentally or over the entire length of the individual selected library member sequence.
- the mutagenized selected library members are then shuffled by in vitro and/or in vivo recombinatorial shuffling as disclosed herein.
- the invention also provides peptide libraries comprising a plurality of individual library members of the invention, wherein (1) each individual library member of said plurality comprises a sequence produced by shuffling of a pool of selected sequences, and (2) each individual library member comprises a variable peptide segment sequence or single-chain antibody segment sequence which is distinct from the variable peptide segment sequences or single-chain antibody sequences of other individual library members in said plurality (although some library members may be present in more than one copy per library due to uneven amplification, stochastic probability, or the like).
- the invention also provides a product-by-process, wherein selected polynucleotide sequences having (or encoding a peptide having) a predetermined binding specificity are formed by the process of: (1) screening a displayed peptide or displayed single-chain antibody library against a predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching library members which bind to the predetermined receptor or epitope to produce a pool of selected library members, (2) shuffling by recombination the selected library members (or amplified or cloned copies thereof) which binds the predetermined epitope and has been thereby isolated and/or enriched from the library to generate a shuffled library, and (3) screening the shuffled library against the predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching shuffled library members which bind to the pre
- the present method can be used to shuffle, by in vitro and or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by antibody display methods, wherein an associated polynucleotide encodes a displayed antibody which is screened for a phenotype (e.g., for affinity for binding a predetermined antigen (ligand)).
- a phenotype e.g., for affinity for binding a predetermined antigen (ligand)
- variable segment genes located upstream of a tandem array of diversity segment genes, which are themselves located upstream of a tandem array of joining (i) region genes, which are located upstream of the constant region genes.
- V-D-J rearrangement occurs wherein a heavy chain variable region gene (VH) is formed by rearrangement to form a fused D segment followed by rearrangement with a V segment to form a V-D-J joined product gene which, if productively rearranged, encodes a functional variable region (VH) of a heavy chain.
- VH heavy chain variable region gene
- VL variable region
- these "affinity sha ⁇ ening" mutations cluster in specific areas of the variable region, most commonly in the complementarity-determining regions (CDRs).
- CDRs complementarity-determining regions
- Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Huse et al, 1989); Caton and Koprowski, 1990; Mullinax et al, 1990; Persson et al, 1991).
- a bacteriophage antibody display library is screened with a receptor (e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled (e.g., to screen plaque or colony lifts).
- a receptor e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid
- immobilized e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography
- labeled e.g., to screen plaque or colony lifts.
- the first step generally involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis.
- the single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3)) or equivalent linker peptide(s).
- the linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH'
- the scfv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site.
- scfv fragments are comprised of VH and VL domains linked into a single polypeptide chain by a flexible linker peptide.
- the scfv genes are assembled, they are cloned into a phagemid and expressed at the tip of the Ml 3 phage (or similar filamentous bacteriophage) as fusion proteins with the bacteriophage PHI (gene 3) coat protein. Enriching for phage expressing an antibody of interest is accomplished by panning the recombinant phage displaying a population scfv for binding to a predetermined epitope (e.g., target antigen, receptor).
- a predetermined epitope e.g., target antigen, receptor
- the linked polynucleotide of a library member provides the basis for replication of the library member after a screening or selection procedure, and also provides the basis for the determination, by nucleotide sequencing, of the identity of the displayed peptide sequence or VH and VL amino acid sequence.
- the displayed peptide (s) or single-chain antibody (e.g., scfv) and/or its VH and VL domains or their CDRs can be cloned and expressed in a suitable expression system.
- polynucleotides encoding the isolated VH and VL domains will be ligated to polynucleotides encoding constant regions (CH and CL) to form polynucleotides encoding complete antibodies (e.g., chimeric or fully-human), antibody fragments, and the like.
- polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework (and optionally constant regions) to form polynucleotides encoding complete antibodies (e.g., humanized or fully-human), antibody fragments, and the like.
- Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography.
- neoplasia a disease that causes inflammation
- neoplasia a malignant neoplasia
- autoimmune disease a malignant neoplasia
- AIDS a malignant neoplasia
- cardiovascular disease a malignant neoplasia .
- infections a malignant neoplasia .
- stage disease e.g., neoplasia
- therapeutic application to treat disease such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.
- VH and VL cassettes can themselves be diversified, such as by random, pseudorandom, or directed mutagenesis.
- VH and VL cassettes are diversified in or near the complementarity-determining regions (CDRS), often the third CDR, CDR3.
- Enzymatic inverse PCR mutagenesis has been shown to be a simple and reliable method for constructing relatively large libraries of scfv site-directed hybrids (Stemmer et al, 1993), as has error-prone PCR and chemical mutagenesis (Deng et al, 1994).
- Riechmann Riechmann et al, 1993
- Barbas (Barbas et al, 1992) attempted to circumvent the problem of limited repertoire sizes resulting from using biased variable region sequences by randomizing the sequence in a synthetic CDR region of a human tetanus toxoid-binding Fab.
- CDR randomization has the potential to create approximately 1 x 10 20 CDRs for the heavy chain CDR3 alone, and a roughly similar number of variants of the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants.
- the combination possibilities of CDR randomization of heavy and/or light chains requires generating a prohibitive number of bacteriophage clones to produce a clone library representing all possible combinations, the vast majority of which will be non-binding. Generation of such large numbers of primary transformants is not feasible with current transformation technology and bacteriophage display systems.
- Intracellular expression of an anti-Rev scfv has been shown to inhibit HIV-1 viras replication in vitro (Duan et al, 1994), and intracellular expression of an anti-p21rar, scfv has been shown to inhibit meiotic maturation of Xenopus oocytes (Biocca et al, 1993). Recombinant scfv, which can be used to diagnose HIV infection have also been reported, demonstrating the diagnostic utility of scfv (Lilley et al, 1994).
- Fusion proteins wherein an scFv is linked to a second polypeptide, such as a toxin or fibrinolytic activator protein, have also been reported (Holvost et al, 1992; Nicholls et al, 1993). [00591] If it were possible to generate scfv libraries having broader antibody diversity and overcoming many of the limitations of conventional CDR mutagenesis and randomization methods, which can cover only a very tiny fraction of the potential sequence combinations, the number and quality of scfv antibodies suitable for therapeutic and diagnostic use could be vastly improved.
- the in vitro and in vivo shuffling methods of the invention are used to recombine CDRs, which have been obtained (typically via PCR amplification or cloning) from nucleic acids obtained from selected displayed antibodies.
- Such displayed antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or any suitable antibody display system wherein the antibody is associated with its encoding nucleic acid(s).
- the CDRs are initially obtained from mRNA (or cDNA) from antibody-producing cells (e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO 92/03918, WO 93/12227, and WO 94/25585), including hybridomas derived therefrom.
- antibody-producing cells e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO 92/03918, WO 93/12227, and WO 94/25585
- Polynucleotide sequences selected in a first selection round are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination, especially shuffling of CDRs (typically shuffling heavy chain CDRs with other heavy chain CDRs and light chain CDRs with other light chain CDRs) to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.
- an antigen e.g., a ligand
- the recombined selected polynucleotide sequences are expressed in a selection format as a displayed antibody and subjected to at least one subsequent selection round.
- the polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection until an antibody of the desired binding affinity is obtained.
- Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral antibody framework sequences (i.e., having insubstantial functional effect on antigen binding), such as for example by back-crossing with a human variable region framework to produce human-like sequence antibodies. Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined antigen.
- the valency of the target epitope may be varied to control the average binding affinity of selected scfv library members.
- the target epitope can be bound to a surface or substrate at varying densities, such as by including a competitor epitope, by dilution, or by other method known to those in the art.
- a high density (valency) of predetermined epitope can be used to enrich for scfv library members, which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity scfv library members.
- a collection of synthetic oligonucleotides encoding random, pseudorandom, or a defined sequence kemal set of peptide sequences can be inserted by ligation into a predetermined site (e.g., a CDR).
- a predetermined site e.g., a CDR
- sequence diversity of one or more CDRs of the single-chain antibody cassette(s) can be expanded by mutating the CDR(s) with site-directed mutagenesis, CDR-replacement, and the like.
- the resultant DNA molecules can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly (i.e., may avoid loss of diversity which may occur upon propagation in a host cell) and the selected library members subsequently shuffled.
- Displayed peptide/polynucleotide complexes which encode a variable segment peptide sequence of interest or a single-chain antibody of interest are selected from the library by an affinity enrichment technique. This is accomplished by means of a immobilized macromolecule or epitope specific for the peptide sequence of interest, such as a receptor, other macromolecule, or other epitope species. Repeating the affinity selection procedure provides an enrichment of library members encoding the desired sequences, which may then be isolated for pooling and shuffling, for sequencing, and/or for further propagation and affinity enrichment.
- the library members without the desired specificity are removed by washing.
- the degree and stringency of washing required will be determined for each peptide sequence or single-chain antibody of interest and the immobilized predetermined macromolecule or epitope.
- a certain degree of control can be exerted over the binding characteristics of the nascent peptide/DNA complexes recovered by adjusting the conditions of the binding incubation and the subsequent washing.
- the temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for nascent peptide/DNA complexes within particular ranges of affinity for the immobilized macromolecule. Selection based on slow dissociation rate, which is usually predictive of high affinity, is often the most practical route.
- nascent peptide/DNA or peptide/RNA complex is prevented, and with increasing time, nascent peptide/DNA or peptide/RNA complexes of higher and higher affinity are recovered.
- affinities of some peptides are dependent on ionic strength or cation concentration. This is a useful characteristic for peptides that will be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptides are required.
- One variation involves the use of multiple binding targets (multiple epitope species, multiple receptor species), such that a scfv library can be simultaneously screened for a multiplicity of scfv which have different binding specificities.
- scfv libraries of as large a size as possible.
- the time and economic considerations of generating a number of very large polysome scFv-display libraries can become prohibitive.
- multiple predetermined epitope species receptor species
- multiple target epitope species each encoded on a separate bead (or subset of beads), can be mixed and incubated with a polysome-display scfv library under suitable binding conditions.
- the collection of beads, comprising multiple epitope species, can then be used to isolate, by affinity selection, scfv library members.
- subsequent affinity screening rounds can include the same mixture of beads, subsets thereof, or beads containing only one or two individual epitope species. This approach affords efficient screening, and is compatible with laboratory automation, batch processing, and high throughput screening methods.
- a variety of techmques can be used in the present invention to diversify a peptide library or single-chain antibody library, or to diversify, prior to or concomitant with shuffling, around variable segment peptides found in early rounds of panning to have sufficient binding activity to the predetermined macromolecule or epitope.
- the positive selected peptide/polynucleotide complexes are sequenced to determine the identity of the active peptides.
- Oligonucleotides are then synthesized based on these active peptide sequences, employing a low level of all bases inco ⁇ orated at each step to produce slight variations of the primary oligonucleotide sequences.
- This mixture of (slightly) degenerate oligonucleotides is then cloned into the variable segment sequences at the appropriate locations.
- This method produces systematic, controlled variations of the starting peptide sequences, which can then be shuffled. It requires, however, that individual positive nascent peptide/polynucleotide complexes be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered complexes and selecting variants having higher binding affinity and/or higher binding specificity.
- mutagenic PCR amplification of positive selected peptide/polynucleotide complexes is done prior to sequencing.
- the same general approach can be employed with single-chain antibodies in order to expand the diversity and enhance the binding affinity/specificity, typically by diversifying CDRs or adjacent framework regions prior to or concomitant with shuffling.
- shuffling reactions can be spiked with mutagenic oligonucleotides capable of in vitro recombination with the selected library members can be included.
- mixtures of synthetic oligonucleotides and PCR produced polynucleotides can be added to the in vitro shuffling mix and be inco ⁇ orated into resulting shuffled library members (shufflants).
- the invention of shuffling enables the generation of a vast library of CDR- variant single-chain antibodies.
- One way to generate such antibodies is to insert synthetic CDRs into the single-chain antibody and/or CDR randomization prior to or concomitant with shuffling.
- the sequences of the synthetic CDR cassettes are selected by referring to known sequence data of human CDR and are selected in the discretion of the practitioner according to the following guidelines: synthetic CDRs will have at least 40 percent positional sequence identity to known CDR sequences, and preferably will have at least 50 to 70 percent positional sequence identity to known CDR sequences.
- a collection of synthetic CDR sequences can be generated by synthesizing a collection of oligonucleotide sequences on the basis of naturally-occurring human CDR sequences listed in Kabat (Kabat et al, 1991); the pool (s) of synthetic CDR sequences are calculated to encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally-occurring human CDR sequence.
- a collection of naturally-occurring CDR sequences maybe compared to generate consensus sequences so that amino acids used at a residue position frequently (i.e., in at least 5 percent of known CDR sequences) are inco ⁇ orated into the synthetic CDRs at the corresponding position(s).
- oligonucleotides encoding CDR peptide sequences encompassing all or most permutations of the observed natural sequence variations is synthesized.
- a collection of human VH CDR sequences have carboxy-terminal amino acids which are either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR oligonucleotide sequences are designed to allow the carboxy-terminal CDR residue to be any of these amino acids.
- residues other than those which naturally-occur at a residue position in the collection of CDR sequences are inco ⁇ orated: conservative amino acid substitutions are frequently inco ⁇ orated and up to 5 residue positions may be varied to inco ⁇ orate non-conservative amino acid substitutions as compared to known naturally-occurring CDR sequences.
- Such CDR sequences can be used in primary library members (prior to first round screening) and/or can be used to spike in vitro shuffling reactions of selected library member sequences. Construction of such pools of defined and/or degenerate sequences will be readily accomplished by those of ordinary skill in the art.
- the collection of synthetic CDR sequences comprises at least one member that is not known to be a naturally-occurring CDR sequence.
- a collection of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, typically at least about 1,000 unique CDR sequences, preferably at least about 10,000 unique CDR sequences, frequently more than 50,000 unique CDR sequences; however, usually not more than about 1 x 10 6 unique CDR sequences are
- Such single-chain antibodies generally bind of about at least 1 x 10 m-, preferably with an affinity of about at least 5 x 10 7 M-l , more preferably with an affinity of at least 1 x 10 8 M-l to 1 x 10 9 M-l or more, sometimes up to 1 x 10 10 M-l or more.
- the predetermined antigen is a human protein, such as for example a human cell surface antigen (e.g., CD4, CD8, TL-2 receptor, EGF receptor, PDGF receptor), other human biological macromolecule (e.g., thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, L-selectin), or nonhuman disease associated macromolecule (e.g., bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.
- a human cell surface antigen e.g., CD4, CD8, TL-2 receptor, EGF receptor, PDGF receptor
- other human biological macromolecule e.g., thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, L-selectin
- nonhuman disease associated macromolecule e.g., bacterial LPS, virion capsid protein or envelope glycoprotein
- variable region encoding sequence may be isolated (e.g., by PCR amplification or subcloning) and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic uses where immunogenicity is preferably minimized.
- the polynucleotide(s) having the resultant fully human encoding sequence(s) can be expressed in a host cell (e.g., from an expression vector in a mammalian cell) and purified for pharmaceutical formulation.
- the antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like (see, generally, Scopes, 1982). Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (see, generally, Lefkovits and Pemis, 1979 and 1981; Lefkovits, 1997).
- the antibodies generated by the method of the present invention can be used for diagnosis and therapy.
- they can be used to treat cancer, autoimmune diseases, or viral infections.
- the antibodies will typically bind to an antigen expressed preferentially on cancer cells, such as erbB-2, CEA, CD33, and many other antigens and binding members well known to those skilled in the art.
- Shuffling can also be used to recombinatorially diversify a pool of selected library members obtained by screening a two-hybrid screening system to identify library members which bind a predetermined polypeptide sequence.
- the selected library members are pooled and shuffled by in vitro and/or in vivo recombination.
- the shuffled pool can then be screened in a yeast two hybrid system to select library members which bind said predetermined polypeptide sequence (e.g., and SH2 domain) or which bind an alternate predetermined polypeptide sequence (e.g., an SH2 domain from another protein species).
- Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell. Intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacz, HIS3) which is operably linked to a Gal4 binding site.
- a reporter gene e.g., lacz, HIS3
- the two-hybrid method is used to identify novel polypeptide sequences which interact with a known protein (Silver and Hunt, 1993; Durfee et al, 1993; Yang et al, 1992; Luban et al, 1993; Hardy et al, 1992; Bartel et al, 1993; and Vojtek et al, 1993).
- variations of the two-hybrid method have been used to identify mutations of a known protein that affect its binding to a second known protein (Li and Fields, 1993; Lalo et al, 1993; Jackson et al, 1993; and Madura et al, 1993).
- Two-hybrid systems have also been used to identify interacting structural domains of two known proteins (Bardwell et al, 1993; Chakrabarty et al, 1992; Staudinger et al, 1993; and Milne and Weaver 1993) or domains responsible for oligomerization of a single protein (Iwabuchi et al, 1993; Bogerd et al, 1993). Variations of two-hybrid systems have been used to study the in vivo activity of a proteolytic enzyme (Dasmahapatra et al, 1992). Alternatively, an E.
- coli/BCCP interactive screening system (Germino et al, 1993; Guarente, 1993) can be used to identify interacting protein sequences (i.e., protein sequences which heterodimerize or form higher order heteromultimers). Sequences selected by a two-hybrid system can be pooled and shuffled and introduced into a two- hybrid system for one or more subsequent rounds of screening to identify polypeptide sequences which bind to the hybrid containing the predetermined binding sequence. The sequences thus identified can be compared to identify consensus sequence(s) and consensus sequence kemals.
- One micro gram samples of template DNA are obtained and treated with U.V. light to cause the formation of dimers, including TT dimers, particularly purine dimers.
- U.V. exposure is limited so that only a few photoproducts are generated per gene on the template DNA sample.
- Multiple samples are treated with U.V. light for varying periods of time to obtain template DNA samples with varying numbers of dimers from U.V. exposure.
- a random priming kit which utilizes a non-proofreading polymerase (for example, Prime-It II Random Primer Labeling kit by Stratagene Cloning Systems) is utilized to generate different size polynucleotides by priming at random sites on templates which are prepared by U.V. light (as described above) and extending along the templates.
- the priming protocols such as described in the Prime-It ⁇ Random Primer Labeling kit may be utilized to extend the primers.
- the dimers formed by U.V. exposure serve as a roadblock for the extension by the non-proofreading polymerase.
- a pool of random size polynucleotides is present after extension with the random primers is finished.
- the invention is further directed to a method for generating a selected mutant polynucleotide sequence (or a population of selected polynucleotide sequences) typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequences(s) possess at least one desired phenotypic characteristic (e.g., encodes a polypeptide, promotes transcription of linked polynucleotides, binds a protein, and the like) which can be selected for.
- a desired phenotypic characteristic e.g., encodes a polypeptide, promotes transcription of linked polynucleotides, binds a protein, and the like
- One method for identifying hybrid polypeptides that possess a desired structure or functional property involves the screening of a large library of polypeptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the polypeptide.
- a predetermined biological macromolecule e.g., a receptor
- the present invention provides a method for generating libraries of displayed polypeptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening.
- the method comprises (1) obtaining a first plurality of selected library members comprising a displayed polypeptide or displayed antibody and an associated polynucleotide encoding said displayed polypeptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequences, optimally introducing mutations into said polynucleotides or copies, (2) pooling the polynucleotides or copies, (3) producing smaller or shorter polynucleotides by interrupting a random or particularized priming and synthesis process or an amplification process, and (4) performing amplification, preferably PCR amplification, and optionally mutagenesis to homologously recombine the newly synthesized polynucleotides.
- the means for blocking or interrapting the amplification or synthesis process is by utilization of UV light, DNA adducts, DNA binding proteins.
- the DNA adducts, or polynucleotides comprising the DNA adducts are removed from the polynucleotides or polynucleotide pool, such as by a process including heating the solution comprising the DNA fragments prior to further processing.
- clones which are identified as having a biomolecule or bioactivity of interest may also be sequenced to identify the DNA sequence encoding a polypeptide (e.g., an enzyme) or the polypeptide sequence itself having the specified activity, for example.
- a polypeptide e.g., an enzyme
- the polypeptide sequence itself having the specified activity for example.
- Suitable clones (e.g., 1-1000 or more clones) from the library are identified by the methods of the invention and sequenced using, for example, high through-put sequencing techniques.
- the exact method of sequencing is not a limiting factor of the invention. Any method useful in identifying the sequence of a particular cloned DNA sequence can be used.
- sequencing is an adaptation of the natural process of DNA replication. Therefore, a template (e.g., the vector) and primer sequences are used.
- One general template preparation and sequencing protocol begins with automated picking of bacterial colonies, each of which contains a separate DNA clone which will function as a template for the sequencing reaction. The selected clones are placed into media, and grown overnight.
- the DNA templates are then purified from the cells and suspended in water. After DNA quantification, high-throughput sequencing is performed using a sequencers, such as Applied Biosystems, Inc., Prism 377 DNA Sequencers. The resulting sequence data can then be used in additional methods, including searching a database or databases.
- a number of source databases are available that contain either a nucleic acid sequence and/or a deduced amino acid sequence for use with the invention in identifying or determining the activity encoded by a particular polynucleotide sequence. All or a representative portion of the sequences (e.g., about 100 individual clones) to be tested are used to search a sequence database (e.g., GenBank, PFAM or ProDom), either simultaneously or individually. A number of different methods of performing such sequence searches are known in the art.
- the databases can be specific for a particular organism or a collection of organisms. For example, there are databases for the C. elegans, Arabadopsis. sp., M.
- sequence data of the clone is then aligned to the sequences in the database or databases using algorithms designed to measure homology between two or more sequences.
- a particular cloned polynucleotide sequence once its identity or activity is determined or a suggested identity or activity is associated with the polynucleotide.
- the desired clone if not already cloned into an expression vector, is ligated downstream of a regulatory control element (e.g., a promoter or enhancer) and cloned into a suitable host cell.
- a regulatory control element e.g., a promoter or enhancer
- Expression vectors are commercially available along with corresponding host cells for use in the invention.
- expression vectors wliich may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, phosmids, bacterial artificial chromosomes, viral nucleic acid (e.g., vaccinia, adenovirus, foul pox viras, pseudorabies and derivatives of SV40), PI -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus, yeast, and the like)
- the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide.
- Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, ⁇ QE-9 (Qiagen), psiX174, pBluescript SK, pBluescript KS, pNH8A, pNHl ⁇ a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia).
- any other plasmid or vector may be used as long as they are replicable and viable in the host.
- the nucleic acid sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
- promoter particularly named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and tip.
- Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviras, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
- the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
- the vector may also include appropriate sequences for amplifying expression. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
- CAT chloramphenicol transferase
- the expression vectors typically contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
- the nucleic acid sequence(s) selected, cloned and sequenced as hereinabove described can additionally be introduced into a suitable host to prepare a library, which is screened for the desired biomolecule or bioactivity.
- the selected nucleic acid is preferably aheady in a vector which includes appropriate control sequences whereby a selected nucleic acid encoding a biomolecule or bioactivity may be expressed, for detection of the desired activity.
- the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
- nucleic acid sequence present in a sample or a particular clone that has been isolated.
- the nucleic acid sequence is amplified by PCR reaction or similar reaction known to those of skill in the art.
- amplification kits are available to carry out such amplification reactions.
- nucleic acid or polypeptide sequences can be implemented in an automated system.
- sequence-based techniques e.g., 96-, 384- and 1536-well .
- the present invention also provides a capillary array- based approach of that has numerous advantages over well-based screening techniques, including the elimination of the need for fluid dispensers for dispensing fluids (e.g., reactants) into individual well reservoirs, and the reduced cost per array (e.g., glass capillaries are reusable) (see, for example, U.S. Patent Application Serial No. 09/444,112, filed November 22, 1999, which is inco ⁇ orated herein by reference in its entirety).
- the capillaries, capillary array and systems of the invention are particularly well suited for screening libraries for activity or biomolecules of interest including polynucleotides.
- the screening for activity may be effected on individual expression clones or may be initially effected on a mixture of expression clones to ascertain whether or not the mixture has one or more specified activities. If the mixture has a specified activity, then the individual clones may be rescreened for such activity or for a more specific activity after collection from the capillary array.
- DNA isolation [00631] DNA is isolated using the IsoQuick Procedure as per manufacture' s instructions (Orca Research Inc., Bothell, WA). The isolated DNA can optionally be normalized according to Example 2 (below). Upon isolation, the DNA is sheared by pushing and pulling the DNA through a 25-gauge double-hub needle and a 1-cc syringe about 500 times. A small amount is run on a 0.8%> agarose gel to make sure the majority of the DNA is in the desired size range (about 3-6kb).
- the DNA is methylated by mixing 4 ⁇ l of 10X EcoRI Methylase Buffer, 0.5 ⁇ l SAM (32 mM), 5.0 ⁇ l EcoRI Methylase (40 u ⁇ l) and incubating at 37 °C for 1 hour. In order to insure blunt ends, the following can be added to the methylation reaction: 5.0 ⁇ l of 100 mM MgCl 2 , 8.0 ⁇ l of dNTP mix (2.5 mM of each dGTP, dATP, dTTP, dCTP), 4.0 ⁇ l of Klenow (5u/ ⁇ l). The mixture is then incubated at 12 °C for 30 minutes.
- the DNA is ligated by gently resuspending the DNA in 8 ⁇ l EcoRI adapters (from Stratagene' s cDNA Synthesis Kit), 1.0 ⁇ l of 10 X ligation buffer, 1.0 ⁇ l of 10 mM rATP, 1.0 ⁇ l of T4 DNA Ligase (4Wu/ ⁇ l) and incubating at 4 °C for 2 days. The ligation reaction is terminated by heating for 30 minutes at 70 °C. [00636] Phosphorylation of adapters .
- the adapter ends are phosphorylated by mixing the ligation reaction with 1.0 ⁇ l of 10X Ligation Buffer, 2.0 ⁇ l of 10 mM rATP, 6.0 ⁇ l of H 2 0, 1.0 ⁇ l of polynucleotide kinase (PNK), and incubating at 37 °C for 30 minutes. After incubating for 30 minutes, 31 ⁇ l of H 2 O and 5 ml of 10 X STE are added to the reaction and the sample is size fractionated on a Sephacryl S-500 spin column. The pooled fractions (1-3) are phenol/chloroform extracted once, followed by an additional chloroform extraction. The DNA is precipitated by the addition of ice cold ethanol on ice for 10 minutes.
- the precipitate is pelleted by centrifugation in a microcentrifuge at high speed for 30 minutes.- The resulting pellet is washed with 1 ml 70% ethanol, repelleted by centrifugation and allowed to dry for 10 minutes. The sample is resuspended in 10.5 ⁇ l TE buffer. The sample is not plated, but is ligated directly to lambda arms as described above, except 2.5 ⁇ l of DNA and no water is used. [ 00637 ] Sucrose Gradient (2.2 ml) Size Fractionation. Ligation is stopped by heating the sample to 65 °C for 10 minutes.
- the sample is gently loaded on a 2.2 ml sucrose gradient and centrifuged in a mini-ultracentrifuged 45k rpm at 20 °C for 4 hours (no brake). Fractions are collected by puncturing the bottom of the gradient tube with a 20 - gauge needle and allowing the sucrose to flow through the needle. The first 20 drops are collected in a Falcon 2059 tube, and then ten 1-drop fractions (labeled 1-10) are collected. Each drop is about 60 ⁇ l in volume. Five ⁇ l of each fraction are run on a 0.8%) agarose gel to check the size.
- Fractions 1-4 (about 10-1.5 kb) are pooled and, in a separate tube, fractions 5-7 (about 5-0.5 kb) are pooled.
- One ml of ice cold ethanol is added to precipitate the DNA and then placed on ice for 10 minutes.
- the precipitate is pelleted by centrifugation in a microcentrifuge at high speed for 30 minutes.
- the pellets are washed by resuspending them in 1 ml of 70% ethanol and repelleting them by centrifugation in a microcentrifuge at high speed for 10 minutes, and then dried. Each pellet is then resuspended in 10 ⁇ l of TE buffer.
- the phage suspension is recovered by pouring the SM buffer off each plate into a 50 ml conical tube. About 3 ml of chloroform are added, shaken vigorously and incubated at room temperature for 15 minutes. The tubes are centrifuged at 2K rpm for 10 minutes to remove cell debris. The supernatant is poured into a sterile flask, 500 ⁇ l chloroform are added and stored at 4 °C.
- the ZAP II library is excised to create the pBLUESCRTPT library according to manufacturer's protocols (Stratagene).
- the DNA library can be transformed into host cells (e.g. , E. coli) to generate an expression library of clones.
- DNA Prior to library generation, purified DNA can be normalized.
- DNA is first fractionated according to the following protocol A sample composed of genomic DNA is purified on a cesium-chloride gradient.
- the DNA is added and thoroughly mixed.
- Ten micrograms of bis-benzimide (Sigma; Hoechst 33258) is added and mixed thoroughly.
- the tube is then filled with the filtered cesium chloride solution and spun in a Bti50 rotor in a Beckman L8-70 Ultracentrifuge at 33k ⁇ m for 72 hours.
- a syringe pump and fractionator (Brandel Model 186) are used to drive the gradient through an ISCO UA-5UV absorbance detector set to 280 nm. Peaks representing the DNA from the organisms present in an environmental sample are obtained. Eubacterial sequences can be detected by PCR amplification of DNA encoding rRNA from a 10 fold dilution of the E. coli peak using the following primers to amplify:
- Recovered DNA is sheared or enzymatically digested to 3-6 kb fragments. Lone- linker primers are ligated and the DNA is size-selected. Size-selected DNA is amplified by PCR, if necessary. [00647] Normalization is then accomplished by resuspending the double-stranded DNA sample in hybridization buffer (0.12 M NaH 2 PO 4 , pH 6.8/0..82 M NaCl/1 mM EDTA/0.1% SDS).
- the sample is overlaid with mineral oil and denatured by boiling for 10 minutes.
- the sample is incubated at 68 °C for 12-36 hours.
- Double-stranded DNA is separated from single-stranded DNA according to standard protocols (Sambrook, 1989) on hydroxyapatite at 60 °C.
- the single-stranded DNA fraction is desalted and amplified by PCR. The process is repeated for several more rounds (up to 5 or more).
- the single plate is grown for 2h at 37 °C and is then used to inoculate two white 96-well Dynatech microtiter daughter plates containing 250 ⁇ l of LB Amp/Meth, glycerol in each well.
- the original single plate is incubated at 37 °C for 18h, then stored at -80 °C
- the two condensed daughter plates are incubated at 37 °C also for 18 h.
- the condensed daughter plates are then heated at 70 °C for 45 min. to kill the cells and inactivate the host E. coli enzymes.
- a stock solution of 5 mg/mL mo ⁇ hourea phenylalanyl-7-amino-4-trifluoromethyl coumarin (MuPheAFC, the "substrate") in DMSO is diluted to 600 ⁇ M with 50 mM pH 7.5 Hepes buffer containing 0.6 mg/mL of the detergent dodecyl maltoside.
- Fifty ⁇ l of the 600 ⁇ M MuPheAFC solution is added to each of the wells of the white condensed plates with one 100 ⁇ l mix cycle using the BIOM ⁇ K to yield a final concentration of substrate of about 100 ⁇ M.
- MuPheAFC The data will indicate whether one of the clones in a particular well is hydrolyzing the substrate.
- the source library plates are thawed and the individual clones are used to singly inoculate a new plate containing LB Amp/Meth, glycerol.
- the plate is incubated at 37 °C to grow the cells, heated at 70 °C to inactivate the host enzymes, and 50 ⁇ l of 600 ⁇ M MuPheAFC is added using the Biomek.
- the enantiomeric excess is determined by either chiral high performance liquid chromatography (HPLC) or chiral capillary electrophoresis (CE). Assays are performed as follows: two hundred ⁇ l of the appropriate buffer is added to each well of a 96-well white microtiter plate, followed by 50 ⁇ l of partially or completely purified enzyme solution; 50 ⁇ l of substrate is added and the increase in fluorescence monitored versus time until 50% of the substrate is consumed or the reaction stops, whichever comes first.
- HPLC high performance liquid chromatography
- CE chiral capillary electrophoresis
- the XLl-Red strain (Stratagene) was transformed with genomic clone 27a3a (in plasmid pBluescript) encoding the alkaline phosphatase gene from the organism OC9a, an organism isolated from the surface of a whale bone, according to the manufacturer's protocol.
- genomic clone 27a3a in plasmid pBluescript
- a 5 ml culture of LB+0.1 mg/ml ampicillin was inoculated with 200 ⁇ l of the transformation and the culture was allowed to grow at 37 °C for 30 hours.
- a miniprep was then performed on the culture, and the isolated DNA screened by transforming 2 ⁇ l of the resulting DNA into XL-1 Blue cells (Stratagene) according to the manufacturer's protocol and following the assay procedure outlined below.
- the mutated OC9a phosphatase took 10 minutes to develop color and the wild type enzyme took 30 minutes to develop color in the screening assay.
- This protocol was used to mutagenize Thermococcus 9N2 Beta-Glycosidase.
- PCR was carried out by incubating 2 microliters dNTP's (10 mM Stocks); 10 microliters lOxPCR Buffer; 0.5 microliters Vector DNA-31G1A-100 nanograms; 20 microliters 3' Primer (100 pmol); 20 microliters 5' Primer (100 pmol); 16 microliters MnCl 4H 2 O (1.25 mM Stock); 24.5 microliters H 2 O; and 1 microliter Taq Polymerase (5.0 Units) in a total volume of 100 microliters.
- the PCR cycle was: 95 °C 15 seconds; 58 °C 30 seconds; 72 °C 90 seconds; 25 cycles (10 minute extension at 72 °C -4°C incubation).
- Five microliters of the PCR product was ran on a 1 % agarose gel to check the reaction. Purify on a QIAQUICK column (Qiagen). Resuspend in 50 microliters H 2 O.
- Twenty-five microliters of purified PCR product 10 microliters NEB Buffer #2; 3 microliters Kpn I (1 OU/microliter); 3 microliters EcoRI (20 U/microliter); and 59 microliters H 2 O.
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CA002456229A CA2456229A1 (en) | 2001-08-03 | 2002-08-05 | Epoxide hydrolases, nucleic acids encoding them and methods for making and using them |
EP02791570A EP1578987A2 (en) | 2001-08-03 | 2002-08-05 | Epoxide hydrolases, nucleic acids encoding them and methods for making and using them |
JP2003517300A JP2005508619A (en) | 2001-08-03 | 2002-08-05 | Epoxide hydrolases, nucleic acids encoding them and methods for making and using them |
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WO2010051288A1 (en) | 2008-10-27 | 2010-05-06 | Revivicor, Inc. | Immunocompromised ungulates |
Also Published As
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US6979733B2 (en) | 2005-12-27 |
JP2005508619A (en) | 2005-04-07 |
CA2456229A1 (en) | 2003-02-13 |
EP1578987A2 (en) | 2005-09-28 |
WO2003012126A3 (en) | 2007-06-14 |
US20030148443A1 (en) | 2003-08-07 |
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