EP1453952A2 - Compositions and methods for normalizing assays - Google Patents
Compositions and methods for normalizing assaysInfo
- Publication number
- EP1453952A2 EP1453952A2 EP02795831A EP02795831A EP1453952A2 EP 1453952 A2 EP1453952 A2 EP 1453952A2 EP 02795831 A EP02795831 A EP 02795831A EP 02795831 A EP02795831 A EP 02795831A EP 1453952 A2 EP1453952 A2 EP 1453952A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- domain
- nucleic acid
- sequence
- intein
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
- C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain
Definitions
- TECHNICAL FIELD This invention generally relates to molecular genetics and pharmaceutical chemistry.
- the invention provides compositions and methods for expression normalization of enzyme activity screens.
- Protein splicing involves the removal of an internal protein sequence from a precursor molecule and the ligation of the two flanking sequences to produce a mature protein product. This post-translational event is analogous to the removal of an intron from rRNA. These protein splicing "introns" have been called “inteins.” The mechanism by which protein splicing is achieved may be entirely encoded within the internal protein sequence, or intein.
- Variability of cell growth and gene expression limits the sensitive detection of changes in enzyme properties. These variations make it difficult to discriminate between changes in gene expression and changes in specific activity in an enzyme activity screen.
- the introduction of expression normalization can improve the sensitivity, reliability and productivity of any activity screen.
- the invention provides a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one cleavage or splicing activity.
- the enzyme is a nitrilase, a racemase, a kinase or a hydroiase, such as epoxide hydroiase, a phosphatase, a lipase or a protease.
- the binding protein is an antibody or a receptor.
- the intein comprises a polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1 , or, the intein comprises a sequence as set forth in SEQ ID NO:2.
- the detectable moiety domain comprises a detectable peptide or polypeptide.
- the detectable peptide or a polypeptide can be a fluorescent peptide or polypeptide.
- the detectable peptide or a polypeptide can be a bioluminescent or a chemiluminescent peptide or polypeptide.
- the bioluminescent or chemiluminescent polypeptide comprises a green fluorescent protein (GFP), an aequorin, an obelin, a mnemiopsin or a berovin.
- the detectable moiety domain comprises an enzyme that generates a detectable signal.
- the enzyme that generates a detectable signal can comprise an alpha-galactosidase, an antibiotic (e.g., chloramphenicol acetyltransferase) or a kinase.
- the detectable moiety domain can comprise a radioactive isotope.
- the chimeric protein is a recombinant fusion protein.
- the intein domain splicing activity results in cleavage of the enzyme domain from the intein domain and detectable domain.
- the intein domain splicing activity can result in cleavage of the enzyme domain from the intein domain and detectable domain and cleavage of the detectable domain from the intein domain.
- the intein domain splicing activity results in cleavage of the detectable domain from the intein domain.
- the intein domain has only splicing activity.
- the intein domain can have only cleaving activity.
- At least one domain is separated from another domain by a linker.
- the linker can be a flexible linker.
- the intein domain can be separated from the detectable moiety domain and the enzyme domain by a linker.
- the invention provides an isolated or recombinant nucleic acid encoding a chimeric protein of the invention, e.g., a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing or cleavage activity.
- the invention provides an expression cassette comprising a nucleic acid of the invention, e.g., an isolated or recombinant nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing or cleavage activity.
- a nucleic acid of the invention e.g., an isolated or recombinant nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein
- the invention provides a vector comprising a nucleic acid of the invention, e.g., an isolated or recombinant nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing activity or one cleaving activity.
- a nucleic acid of the invention e.g., an isolated or recombinant nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or
- the invention provides a cell comprising a nucleic acid of the invention, e.g., a nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing or cleavage activity.
- the invention provides a non-human transgenic animal comprising a nucleic acid of the invention, e.g., a nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing or cleavage activity.
- a nucleic acid of the invention e.g., a nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable mo
- the invention provides a method for normalizing gene expression comprising the following steps: (a) providing a nucleic acid encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing or cleavage activity; (b) expressing the nucleic acid such that the chimeric protein is expressed and the intein domain has at least one splicing activity; and (c) measuring both the activity of the enzyme or the binding protein and the amount of detectable moiety domain expressed, thereby normalizing gene expression.
- the detectable moiety domain comprises a detectable peptide or polypeptide.
- the detectable peptide or a polypeptide can be a fluorescent peptide or polypeptide.
- the fluorescent peptide or polypeptide can be expressed in a cell and the fluorescence can be measured by a FACS or equivalent device.
- the nucleic acid is expressed in vivo, or, the nucleic acid is expressed in vitro.
- the invention provides a high throughput enzymatic screen to measure enzyme activity comprising the following steps: (a) providing a nucleic acid or expression vehicle (e.g., a vector) of the invention, e.g., encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing or cleavage activity; (b) expressing the nucleic acid or expression vehicle (e.g., a vector) in vivo or in vitro; and (c) measuring both the activity of the enzyme and the amount of detectable moiety domain expressed.
- a nucleic acid or expression vehicle e.g., a vector
- the nucleic acid or expression vehicle e.g
- the invention provides a high throughput binding protein screens to measure binding activity comprising the following steps: (a) providing a nucleic acid or expression vehicle (e.g., a vector) of the invention, e.g., encoding a chimeric protein comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one splicing or cleavage activity; (b) expressing the nucleic acid or expression vehicle (e.g., a vector) in vivo or in vitro; and (c) measuring both the activity of the binding protein and the amount of detectable moiety domain expressed.
- the invention provides an isolated or recombinant polypeptide comprising
- a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:2 over a region of at least about 100 residues or (b) a polypeptide encoded by a nucleic acid comprising (i) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:l over a region of at least about 100 residues; or, (ii) a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a sequence as set forth in SEQ ID NO: 1, or a subsequence thereof, wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection and the polypeptide has an intein activity.
- the intein activity is a cleaving activity or a splicing activity.
- the polypeptide can comprise (or consisting of) an amino acid sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more sequence identity to SEQ ID NO:2 over a region of at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or more residues.
- the polypeptide can have the amino acid sequence as set forth in SEQ ID NO:2.
- the isolated or recombinant intein polypeptide is not associated with any sequence to which it is naturally associated (e.g., an Aquifex sequence) on its amino terminal end, on its carboxy terminal end, or on both ends of the intein.
- the invention provides an isolated or recombinant polypeptide comprising (or consisting of) a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:2 over a region of at least about 100 residues with the proviso that it is not associated with any sequence to which it is naturally associated (e.g., an Aquifex sequence) on its amino terminal end, on its carboxy terminal end, or on both ends of the intein.
- the invention provides an isolated or recombinant nucleic acid comprising
- nucleic acid (or consisting of) a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:l over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- the nucleic acid can comprise a sequence having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%, 98%, 99%, or more sequence identity to SEQ ID NO:l over a region of at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900 or 1000 or more residues.
- 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 isolated or recombinant nucleic acid is not associated with any sequence to which it is naturally associated (e.g., an Aquifex sequence) on its 3' terminal end, on its 5' terminal end, or on both ends of the intein coding sequence.
- the invention provides an isolated or recombinant nucleic acid comprising (or consisting of) a sequence having at least 90% identity to SEQ ID NO: 1 over a region of at least about 100 residues with the proviso that it is not associated with any sequence to which it is naturally associated (e.g., an Aquifex sequence) on its 3' end, on its 5' end, or on both ends of the intein-coding sequence.
- 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: 1.
- the nucleic acid is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900 or 1000 or more residues in length or the full length of the gene or transcript.
- the stringent conditions can 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 intein activity, wherein the probe comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or more consecutive bases of a sequence comprising a sequence as set forth in SEQ ID NO:l.
- the invention provides an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having an intein activity, wherein the primer pair is capable of amplifying a nucleic acid comprising a sequence as set forth in SEQ ID NO: 1.
- the invention provides a method of amplifying a nucleic acid encoding a polypeptide having an intein activity comprising amplification of a template nucleic acid with an amplification primer sequence pair capable of amplifying a nucleic acid sequence as set forth in SEQ ID NO: 1, or a subsequence thereof.
- the invention provides an expression cassette comprising a nucleic acid of the invention, e.g., a sequence having at least 90% sequence identity to SEQ ID NO:l over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- a nucleic acid of the invention e.g., a sequence having at least 90% sequence identity to SEQ ID NO:l over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- the invention provides a vector comprising a nucleic acid sequence of the invention, e.g., a nucleic acid having at least 90% sequence identity to SEQ ID NO: 1 over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- the invention provides a cloning vehicle comprising a vector of the invention, e.g., as set forth in claim 65, 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 invention provides a transformed cell comprising an expression cassette of the invention or a vector of the invention.
- the cell can be a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or
- the invention provides a transgenic non-human animal comprising a nucleic acid of the invention, e.g., a sequence having at least 90% sequence identity to SEQ ID NO: 1 over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- a nucleic acid of the invention e.g., a sequence having at least 90% sequence identity to SEQ ID NO: 1 over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- the invention provides a transgenic plant comprising a nucleic acid of the invention, e.g., a sequence having at least 90% sequence identity to SEQ ID NO:l over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- a nucleic acid of the invention e.g., a sequence having at least 90% sequence identity to SEQ ID NO:l over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- the invention provides a method of inhibiting the translation of an intein 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 sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid having at least 90% sequence identity to SEQ ID NO: 1 over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.
- an antisense oligonucleotide comprising a nucleic acid of the invention, e.g., a sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid having at least 90% sequence identity to SEQ ID NO: 1 over a region of at least about 100 residues, wherein the nucleic acid encodes at least one polypeptide having an intein activity, and
- the invention provides an isolated or recombinant antibody that specifically binds to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
- Figure 2 is a diagram of the constructs used to generate exemplary intein expression vectors, as set forth in detail in Example 1, below.
- Figure 3 is a schematic diagram of splicing versus cleavage of the Aquifex aeolicus intein, as set forth in detail in Example 1, below.
- Figure 4 is an illustration whole cell extracts as analyzed by SDS-PAGE, as set forth in detail in Example 1, below.
- Figure 5 illustrates the overexpression of NMA intein in BL21 (DE3)RIL, as set forth in detail in Example 1, below.
- Figure 6 illustrates exemplary chimeric structures that retain intein function and fluorescence, as set forth in detail in Example 1, below.
- Figure 6A illustrates flexible linkers introduced between the intein and FP domains.
- Figure B illustrates fusion of the GFP C-terrninal to the intein, as set forth in detail in Example 1 , below.
- Figure 7 illustrates ⁇ -gal fusions to NMA-FP inteins, as set forth in detail in Example 1, below.
- Figure 8 schematically illustrates an exemplary method of the invention for making libraries of inteins using GeneReassemblyTM.
- Figure 8A shows recombination locations between essential splicing motifs.
- Figure 8B schematically illustrates an exemplary method of making a complex library of all possible combinations of splicing motifs, as set forth in detail in Example 1, below.
- Figure 9 illustrates an exemplary method of the invention where at the same time that the intein deletions are screened a variety of linkers can be added to the ⁇ - and/or C- terminal of the detectable protein, as set forth in detail in Example 1 , below.
- Figure 10 schematically illustrates an exemplary method of the invention that creates a library in intein mutants using gene mutation (GSSM) and gene reassembly, antibiotic selection with kanamycin followed by kanamycin resistant activity assays to generate inteins with increased splicing efficiency, as set forth in detail in Example 1, below.
- GSSM gene mutation
- Figure 11 schematically illustrates an exemplary method of the invention that creates a library in intein mutants using gene mutation (GSSM) and gene reassembly, antibiotic selection with kanamycin followed by kanamycin resistant activity assays to generate inteins with increased cleavage efficiency, wherein the intein is fused between the target gene as an "N-extein” and a His6 affinity tag as a "C-extein,” as set forth in detail in Example 1, below.
- GSSM gene mutation
- Figure 12 schematically illustrates an exemplary method of the invention to quantitate the enantioselective conversion of amino acids using secondary detection enzymes, as set forth in detail in Example 1, below.
- Figure 13 schematically illustrates an exemplary nitrilase activity assay of the invention using the fluorogenic reagent dihydroxyphenoxazine, as set forth in detail in Example 1, below.
- Figure 14 schematically illustrates exemplary assays of the invention: a chromogenic epoxide assay and a fluorogenic epoxide assay, in Figure 14A and Figure 14B, respectively.
- the invention provides compositions and methods for expression normalization of enzyme activity screens.
- the invention provides chimeric polypeptides ("fusion proteins") with at least three domains: a first domain comprising at least one enzyme, a second domain between the first and the third domain comprising at least one intein and a third domain comprising at least one detectable moiety (domain), e.g., a protein such as a fluorescent domain or a bioluminescent protein, or any other detectable moiety.
- the chimeric polypeptide of the invention is expressed recombinantly in vitro or in a cell, i.e., in vivo. After expression of the protein the cleavage activity of the intein moiety produces a stoichiometric expression of the two proteins, i.e., the enzyme of the first domain and the detectable moiety of the third domain.
- the chimeric polypeptides of the invention can be used for expression normalization of enzyme activity, e.g., in assays and screens.
- the compositions and methods of the invention can improve the sensitivity, reliability and productivity of any activity screen.
- the compositions and methods of the invention can be used in high throughput enzymatic screens to measure enzyme activities in whole cells or cell lysates.
- compositions and methods of the invention can be used to monitor enzymatic conversions directly or indirectly in a single cell assay, e.g., a FACS-based assay, or in a group of cells, e.g., those grown in a well of a microtiter plate.
- the compositions and methods of the invention can be used to discriminate high specific activity enzymes, e.g., clones, from high expressing enzymes, e.g., clones, even though different genes (or enzyme coding sequences in vectors) can be expressed at dramatically different levels.
- the compositions and methods of the invention can be used to decrease background noise generated by liquid transfer errors and differences in growth and expression.
- compositions and methods of the invention are used to normalize enzyme activity to gene expression to improve the sensitivity and reliability of an assay, e.g., an enzyme assay.
- an assay e.g., an enzyme assay.
- the compositions and methods of the invention can be highly sensitive, in one aspect they are used to develop new assays, e.g., enzyme assays, that otherwise might have been impossible due to variations in gene expression.
- the improved sensitivity allows detection of small activity increases, e.g., the small activity increases that are generated in each cycle of an iterative enzyme evolution. By themselves, small increases in enzyme activity may not significantly improve the value of an enzyme. However, the synergy generated by combining many small mutations iteratively can result in very dramatic improvements in enzyme activity.
- the fluorescent measurement of gene expression increases the sensitivity and information generated by standard microtiter plate screens for enzyme activity.
- the screening methods of the invention can completely cover large enzyme libraries, e.g., for enzyme discovery and evolution.
- the invention provides sub-microliter and single cell assays to detect enzyme activity for discovery purposes.
- the invention provides quantitative activity measurements for, e.g., detecting desirable mutants and for enzyme evolution activity assays.
- the methods normalize expression (e.g., enzyme expression) to fluorescence.
- the compositions and methods of the invention can be used for ultra high throughput enzyme screens. In some aspects, this improves throughput by several orders of magnitude and can enable complete sampling of the enzyme libraries.
- compositions and methods of the invention can be used in any assay, including enzymatic screens and spectroscopic assays.
- the compositions and methods of the invention are used in spectroscopic assays, e.g., in microtiter plates, GigaMatrixTM, and FACS.
- activity is measured by a variety of biological and analytical methods. All assays providing a quantitative value for activity are incorporated into the methods of the invention to normalize activity to protein expression.
- the methods of the invention are used as a general technique to achieve stoichiometric expression of two proteins without the limitations of a fusion protein.
- the intein domain of a chimeric polypeptide of the invention e.g., an intein-fluorescent protein
- intein domain of a chimeric polypeptide of the invention can function equally well in eukaryotic as well as prokaryotic systems.
- compositions and methods of the invention can be applied to any organism.
- the chimeric polypeptides of the invention can be used to develop, e.g., "evolve," enzymes.
- the chimeric polypeptides of the invention can be used to "evolve" enzymes for the biocatalytic production of commercial products, e.g., pharmaceuticals and industrial chemicals and proteins.
- Any enzyme can be linked to a detectable domain via an intein, e.g., a hydroiase, a racemase, a nitrilase and/or a epoxide hydroiase enzyme.
- Targets of racemases can be proteogenic or non-proteogenic amino acids.
- the intein domain of a chimeric (multi-domain) protein of the invention has more cleavage activity than splicing activity.
- the intein domain has greater than 50%, 60%, 70%, 80%, 90%, 95% or 98% or more in vivo ox in vitro splicing activity.
- a target protein e.g., a substrate
- a purified form e.g., a substrate
- high expressing genes identified.
- the invention provides nucleic acids encoding the chimeric polypeptides of the invention. Also provided are expression systems, e.g., expression cassettes, recombinant viruses, vectors, and the like for expressing and using the chimeric proteins of the invention in enzyme activity assays.
- the coding sequences can be operably linked to a transcriptional control sequence, such as a promoter, e.g., a constitutive or an inducible promoter.
- intein includes all polypeptides capable of any intein activity, e.g., capable of protein splicing or protein cleaving involving the removal of an internal protein sequence. In one aspect, the intein is also capable of ligating the two flanking sequences.
- the invention provides expression cassettes comprising nucleic acids of the invention.
- expression cassette includes nucleotide sequences which are capable of affecting expression of a coding sequence (e.g., a chimeric 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.
- expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
- a "vector” can comprise 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 include both the expression and non-expression plasmids.
- RNA autonomous self-replicating circular or linear DNA or RNA
- plasmids viruses, and the like, see, e.g., U.S. Patent No. 5,217,879
- the invention provides nucleic acids operably linked to a coding sequence of the invention.
- "Operably linked” as used herein can refer to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory sequence to a transcribed sequence.
- a promoter is operably linked to a coding sequence, such as a nucleic acid of the invention, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
- promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
- promoter can include all sequences capable of driving transcription of a coding sequence in a cell, e.g., a plant cell.
- promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene.
- a promoter can be a c ⁇ -acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cz ' s-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
- Constutive promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation.
- “Inducible” or “regulatable” promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.
- nucleic acid or “nucleic acid sequence” can include 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).
- the term encompasses 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) Biochem. 36:8692-8698; Straussense Nucleic Acid Drug Dev 6:153-156.
- isolated can include a material 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 invention 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.
- nucleic acids can include nucleic acids adjacent to a “backbone” nucleic acid to which 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.
- the enriched nucleic acids represent 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% 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.
- amino acid or “amino acid sequence” can include 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 include 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 invention also include all “mimetic” and “peptidomimetic” forms, as described in further detail, below. Generating and Manipulating Nucleic Acids
- the invention provides nucleic acids, including expression cassettes such as expression vectors, encoding the chimeric polypeptides of the invention.
- the invention also includes methods for discovering new chimeric and new intein sequences using the nucleic acids of the invention. Also provided are methods for modifying the nucleic acids of the invention, including the intein 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.
- nucleic acids used to practice the methods of the invention e.g., enzymes, binding proteins and/or inteins
- 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;
- MACs mammalian artificial chromosomes
- human artificial chromosomes see, e.g., Rosenfeld (1997) Nat. Genet. 15:333- 335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); PI artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; Pl-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids.
- YAC yeast artificial chromosomes
- BAC bacterial artificial chromosomes
- PI artificial chromosomes see, e.g., Woon (1998) Genomics 50:306-316
- Pl-derived vectors see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids.
- 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 chimeric polypeptide of the invention can be further fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
- the chimeric polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., 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 tip.
- 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, e.g., the E.
- Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 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.
- the invention provides expression vehicles comprising the chimeric compositions of the invention. These expression vehicles can be used in various screening assays.
- the nucleic acids of the invention comprise gene libraries, e.g., for screening for various intein constructs, enzyme domains or binding protein domains, detectable moiety domains, selection markers and the like. Different vectors may be needed to suit the specific needs of different applications.
- the nucleic acids or expression vehicles (e.g., vectors) of the invention can comprise appropriate multiple cloning sites for fusing genes and/or libraries C-terminal to the intein-detectable domain fusion (e.g., intein-FP).
- the nucleic acids or expression vehicles (e.g., vectors) of the invention can comprise promoters for inducible and/or constitutive expression of genes.
- the nucleic acids or expression vehicles (e.g., vectors) of the invention can be compatible with different screening hosts, such as bacilli, e.g., E. coli, Pseudomonas, Streptomyces, Bacillus, etc., yeast, insect, plant, mammalian and the like.
- the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the inteins 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), P 1 -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.
- exemplary vectors are include: bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda- ZAP vectors (Stratagene); ptrc99a, pKK223-3, ⁇ DR540, ⁇ RIT2T (Pharmacia); Eukaryotic: pXTl, pSG5 (Stratagene), ⁇ SVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).
- 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 can 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 TRP1 gene.
- Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers.
- CAT chloramphenicol transferase
- Enhancers are cis-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.
- Particular bacterial vectors which can 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) pQE70, pQE60, pQE-9 (Qiagen), pDIO, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia), pKK232-8 and pCM7.
- Particular eukaryotic vectors include pSV2CAT, pOG44, pXT 1 , pSG (Stratagene) pS VK3 , pBPV, pMSG, and pSVL (Pharmacia).
- any other vector may be used as long as it is replicable and viable in the host cell.
- the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses and transiently or stably expressed in plant cells and seeds.
- One exemplary transient expression system uses episomal expression systems, e.g., cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus by transcription of an episomal mini-chromosome containing supercoiled DNA, see, e.g., Covey (1990) Proc. Natl. Acad. Sci. USA 87:1633-1637.
- coding sequences, i.e., all or sub-fragments of sequences of the invention can be inserted into a plant host cell genome becoming an integral part of the host chromosomal DNA.
- Sense or antisense transcripts can be expressed in this manner.
- a vector comprising the sequences (e.g., promoters or coding regions) from nucleic acids of the invention can comprise a marker gene that confers a selectable phenotype on a plant cell or a seed.
- the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.
- the invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding an intein 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. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477, U.S. Patent No. 5,750,870.
- the vector can 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)).
- Cell-free translation systems can also be employed to produce a chimeric 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 kanamycin, 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 kanamycin, tetracycline or ampicillin resistance in E. coli.
- nucleic acids of the invention and nucleic acids encoding the polypeptides of the invention, or modified nucleic acids of the invention can be reproduced by amplification.
- Amplification can also be used to clone or modify the nucleic acids of the invention.
- the invention provides amplification primer sequence pairs for amplifying nucleic acids of the invention.
- the primer pairs are capable of amplifying nucleic acid sequences including the exemplary SEQ ID NO:l, or a subsequence thereof
- the exemplary SEQ ID NO:l is atggaaaaga cagaaaaaaa tgagcttgtc agaaaactca ttttcaaccc tcaaggagac 60 agggaggcga gcaaaggaa gataataaag ggaaacccga caaacatatt tgaacttaac 120 gagataaagt attcctgggc ttttgacctt tacaagttaa tgggctttac aaacttctgg 180 atacccgaag agatacagat gcttgaagac aggaaacagt acgagaccgt tctatcagac 240 tacgaaaaga gggcatacga actcgtcctttttctca tagcccttga ctttttttttt
- an exemplary amplification primer sequence pair is residues 1 to 21 of SEQ ID NO: 1 (atggaaaaga cagaaaaaa t) and the complementary strand of the last 21 residues of SEQ ID NO:l (gccaacgaac tcaagtggta a).
- Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an array or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample.
- message isolated from a cell or a cDNA library are amplified.
- Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- transcription amplification see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173
- self-sustained sequence replication see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874)
- Q Beta replicase amplification see, e.g., Smith (1997) J. Clin. Microbiol.
- the invention provides nucleic acids having at least 90% sequence identity to SEQ ID NO: 1.
- 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 ID NO: 1.
- the sequence identity can be over a region of at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, or more consecutive residues, or the full length of the nucleic acid or polypeptide.
- 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,
- 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 (a sequence of the invention, e.g., SEQ ID NO:l.
- a "comparison window", as used herein, includes reference to a segment of any one of the numbers of contiguous residues.
- contiguous residues ranging anywhere from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- reference sequence has the requisite sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, e.g., 50%, 55%, 60%, 65%, 70%, 15%, 80%, 90% or 95% sequence identity to a sequence of the invention, including SEQ ID NO: 1.
- various subsequences 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.
- 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.
- Word Size 3 Matrix: Blosum62 Gap Costs: Existence: 11 Extension: 1"
- Other default settings can be: filter for low complexity OFF, word size of 3 for protein, BLOSUM62 matrix, gap existence penalty of -11 and a gap extension penalty of -1.
- An exemplary NCBI BLAST 2.2.2 program setting has the "-W" option default to 0. This means that, if not set, the word size defaults to 3 for proteins and 11 for nucleotides.
- 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.
- the invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention, e.g., a sequence as set forth in SEQ ID NO:l, or a nucleic acid that encodes a polypeptide of the invention, e.g., SEQ ID NO:2.
- the stringent conditions can be highly stringent conditions, medium stringent conditions, low stringent conditions, including the high and reduced stringency conditions described herein. In one aspect, it is the stringency of the wash conditions that set forth the conditions which determine whether a nucleic acid is within the scope of the invention, as discussed below.
- 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 nucleic acid 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, or more, residues in length. ' Nucleic acids shorter than full length are also included. These nucleic acids can be 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 acids of the invention are defined by their ability to hybridize under high stringency comprises conditions of about 50% formamide at about 37°C to 42°C. In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30°C to 35°C.
- nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA).
- nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35°C. Following hybridization, 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.
- a specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide.
- a specific example of "low stringency” hybridization conditions is when the above hybridization is conducted at 10% formamide.
- the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
- Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions are well known in the art. Hybridization conditions are discussed further, below.
- 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 may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na + concentration of approximately 1M.
- the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization.
- “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.
- 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 0J5 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 0JX SSC containing 0.1% SDS at 68oC for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen and Ausubel for
- the invention also provides nucleic acid probes that can be used, e.g., for identifying nucleic acids encoding an intein of the invention.
- the probe comprises at least 10 consecutive bases of a nucleic acid of the invention.
- a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention.
- the probes identify a nucleic acid by binding and/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).
- the invention provides methods for mutating a nucleic acid sequence of the invention, including an intein of the invention.
- the methods comprise providing segments of a chromosome or part of a chromosome and introducing one or more mutations into one or more of the segments. Any method can be used to introduce the mutation, either randomly, non-randomly, or both. These methods can be repeated or used in various combinations to generate one or more mutations into one or more of the segments. These methods also can be repeated or used in various combinations.
- the genetic composition of a cell is altered by, e.g., modification of a homologous gene ex vivo by a method of the invention followed by its reinsertion into a cell.
- Any method can be used to introduce the mutation, either randomly, non- randomly, or both.
- 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.
- 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.
- 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-aminopurine, or acridine.
- 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 mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation
- 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 (1985) "In vitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985) "Strategies and applications of in vitro mutagenesis” Science 229:1193-1201; Carter (1986) "Site-directed mutagenesis” Biochem. J.
- 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. Acids Res.
- 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 mutated nucleic acid segments.
- Polypeptides encoded by these 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.
- non-stochastic gene modification a "directed evolution process” is used to generate mutated sequences. 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.
- 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.
- 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 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 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.
- 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. For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3 x 3 x 3 or 27 total possibilities, including 7 that were previously examined - 6 single point mutations (i.e. 2 at each of three positions) and no change at any position.
- 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.
- This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.
- Synthetic Ligation Reassembly The methods of the invention include use of non-stochastic gene modification system termed "synthetic ligation reassembly," or simply “SLR,” a “directed evolution process,” to generate mutated sequences of the invention.
- 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.
- 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 co ⁇ esponding 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 methods of the invention also use non-stochastic gene modification system termed "optimized directed evolution system" to generate mutated sequences of the invention, e.g., modified inteins and chimeric polypeptide coding sequences.
- 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.
- this method provides a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.
- Previously if one generated, for example, 10 13 chimeric molecules during a reaction, it would be extremely difficult to test such a high number of chimeric variants for a particular activity.
- a significant portion of the progeny population would have a very high number of crossover events which resulted in proteins that were less likely to have increased levels of a particular activity.
- 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.
- One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides co ⁇ esponding 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 co ⁇ ect 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 co ⁇ esponding 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 each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides co ⁇ esponding 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 co ⁇ ect 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.
- 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 co ⁇ esponding 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 co ⁇ ect 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 co ⁇ esponding 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 each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides co ⁇ esponding 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 processes can be iteratively repeated. For example a nucleic acid (or, the nucleic acid) responsible for an intein activity is identified, re-isolated, again modified, re-tested for activity. This process can be iteratively repeated until a desired intein phenotype is engineered. An entire biochemical anabolic or catabolic pathway can be engineered into a cell, including proteolytic activity. Similarly, if it is determined that a particular oligonucleotide has no affect at all on the desired trait, it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed.
- In vivo shuffling of molecules can be used in methods of the invention, e.g., to develop modified inteins and chimeric polypeptides.
- 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.
- hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules.
- hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.
- the methods of the invention introduce one or more mutations into one or more of nucleic acid segments, e.g., inteins and chimeric sequences of the invention.
- the nucleic acids can be altered by any means, including, e.g., random or stochastic methods, or, non-stochastic, or "directed evolution," methods, as described above.
- Mutations can 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.
- nucleotide differences can result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
- mutations may be created using e ⁇ or prone PCR.
- e ⁇ or 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.
- E ⁇ or 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, MgCl 2 , MnCl 2 , 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 50 mM KC1, 10 mM Tris HC1 (pH 8.3) and 0.01% gelatin, 7 mM MgCl 2 , 0.5 mM MnCl 2 , 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM 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. Mutations 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.
- 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 mutations 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 perfo ⁇ ned by resuspending the purified fragments at a concentration of 10-30 ng/:l in a solution of 0.2 mM of each dNTP, 2.2 mM MgCl 2 , 50 mM KCL, 10 mM Tris HCl, 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. Mutations 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.
- Mutations 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 mutations.
- 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.
- 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) Cu ⁇ ent Opinion in Biotechnology 4:450-455.
- mutations 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 provides methods for modifying intein-encoding nucleic acids (and the nucleic acids encoding the chimeric polypeptides of the invention) to modify codon usage.
- the invention provides methods for modifying codons in a nucleic acid encoding an intein to increase or decrease its expression in a host cell.
- the invention also provides nucleic acids encoding an intein modified to increase its expression in a host cell, intein so modified, and methods of making the modified inteins.
- the method comprises identifying a "non-prefe ⁇ ed” or a "less prefe ⁇ ed” codon in intein- encoding nucleic acid and replacing one or more of these non-prefe ⁇ ed or less prefe ⁇ ed codons with a "prefe ⁇ ed codon” encoding the same amino acid as the replaced codon and at least one non-prefe ⁇ ed or less prefe ⁇ ed codon in the nucleic acid has been replaced by a prefe ⁇ ed codon encoding the same amino acid.
- a prefe ⁇ ed codon is a codon over- represented in coding sequences in genes in the host cell and a non-prefe ⁇ ed or less prefe ⁇ ed 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, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines.
- the invention also includes nucleic acids and polypeptides optimized for expression in these organisms and species.
- the codons of a nucleic acid encoding an intein or a chimeric polypeptide of the invention 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 intein 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.
- the invention provides transgenic non-human animals comprising a nucleic acid (e.g., encoding an intein or a chimeric polypeptide of the invention), a polypeptide, an expression cassette or vector or a transfected or transformed cell of the invention.
- the invention also provides methods of making and using these transgenic non-human animals.
- 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 intein activity, or, as models to screen for agents that change the intein 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 disruption of the gene encoding amyloid precursor protein (APP).
- APP amyloid precursor protein
- the invention provides chimeric polypeptides comprising at least three domains, wherein the first domain comprises at least one enzyme domain or a binding protein domain, the second domain comprises at least one intein domain and a third domain comprising a detectable moiety domain, at least one intein domain is positioned between at least one enzyme or binding protein and at least one detectable moiety domain, and the intein domain has at least one cleavage or splicing activity.
- the invention also provides isolated or recombinant polypeptides having a sequence identity to an exemplary sequence of the invention, e.g., SEQ ID NO:2: Met Glu Lys Thr Glu Lys Asn Glu Leu Val Arg Lys Leu He Phe Asn 1 5 10 15
- 515 520 525 lie Tyr Leu Glu Asp Asp Tyr He Asn Thr Gin Thr Met Val Lys Glu
- FP fluorescent proteins
- they are compatible with other fluorescent assays, e.g., a variety of colors (green, cyan, red and yellow) can be used. The variety of colors provides flexibility in choosing a FP with spectroscopic activity that does not overlap with that of the enzyme domain or binding protein domain (e.g., the target enzyme).
- Polypeptides and peptides 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., Caruthers (1980) Nucleic Acids Res. Symp. Ser.
- peptide synthesis can be performed using various solid-phase techmques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis may be 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 incorporates 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 include all “mimetic” and “peptidomimetic” forms.
- the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound which has substantially the same structural 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 incorporate 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.
- a mimetic composition is within the scope of the invention if it has an intein 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 structural 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 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.
- peptide bonds 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).
- DCC N,N'-dicyclohexylcarbodiimide
- DIC N,N'- diisopropylcarbodiimide
- 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-pyridinyl)-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-biphenyl- phenylalanine; D- or L-p-methoxy-bipheny
- Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, py ⁇ olyl, 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 ornithine, citrulline, 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 co ⁇ esponding 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 co ⁇ esponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
- 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 refe ⁇ ed 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 structural type or a peptidomimetic, but of the opposite chirality, refe ⁇ ed to as the D- amino acid, but also can be refe ⁇ ed to as the R- or
- 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. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., pp. 11-12)) and have recently been employed in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally utilized the teachings of H. M. Geysen et al, . Proc. Natl. Acad.
- 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.
- 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.
- the chimeric compositions of the invention can comprise any known intein, of which there is a wide range of sequence diversity, splicing behavior, and structures.
- Existing inteins comprise a large family, see, e.g., Perler (2000) Nucleic Acids Res. 28:344-345.
- the inteins used in the methods of the invention, as part of the chimeric compositions of the invention, including the inteins of the invention, can be recombinantly generated, synthetic, isolated or a combination thereof.
- the invention provides an isolated intein of 346 amino acids. This intein was isolated the wild type Aquifex. It has a number of rare codons. Methods for modifying the expression of inteins
- the invention provides methods for modifying the function and expression of inteins, including the inteins of the invention.
- the methods can comprise any protocol for modifying nucleic acid and/or polypeptide sequences and/or structures.
- the methods of the invention comprise the use of GSSMTM, or Gene Site Saturation MutagenesisTM.
- GSSM is a method of systematically generating every possible amino acid mutation at every residue of a particular gene.
- intein mutagenesis focuses on intein regions responsible for splicing.
- the intein-modifying procedures develop an intein that has little or no affect on gene expression.
- an intein of the invention has little or no affect on gene expression when fused to a target gene.
- the intein-modifying procedures identify the minimal intein regions necessary for optimal intein expression without affecting splicing activity.
- the relatively small domains at the N- and C-terminus of the intein are required for splicing activity; see, e.g., ., Chong (1997) J. of Biol. Chem. 272:15587- 15590; Telenti (1997) J. of Bacteriol. 179:6378-6382; Lew (1998) J. of Biol. Chemistry 273:15887-15890.
- the invention provides intein variants where sequences not responsible for activity, e.g., splicing activity, are removed.
- the deleted intein sequences are replaced with another sequence, e.g., a protein coding sequence, such as a detectable protein, e.g., a fluorescent protein (FP).
- FP fluorescent protein
- the invention provides an intein whose non-splicing sequences have been replaced with a detectable protein (e.g., a fluorescent protein).
- this sequence is placed in an expression system, e.g., a vector.
- this chimeric sequence is expressed and used to normalize gene expression.
- linkers are added to the N- and/or C- terminal of the detectable protein, e.g., FP.
- coding sequences for linkers are added to the N- and/or C- terminal of the coding sequence of the detectable protein.
- a variety of linkers can be added to the N- and/or C- terminal of the detectable protein, e.g., FP; see Figure 9.
- coding sequences for a variety of linkers can be added to the coding sequences of the N- and/or C- terminal of the detectable protein, e.g., FP.
- the minimal intein constructs are screened for function (e.g., splicing, enzymatic activity) in the pEKIl construct (pEKintl, see Figure 2). In one aspect, this only provides kanamycin resistance if the intein is able to splice.
- a large number of intein/linker combinations are screened to find a minimal intein that retains splicing and FP function (see Figure 9).
- this "mini-intein” expresses at higher levels when fused to a target protein.
- an intein used in the compositions and/or methods of the invention is modified by optimizing its codon usage for high expression in various screening hosts, e.g., yeast, bacteria and the like. Codon optimization of a minimal intein may be easier than of a full-length intein. In one aspect, these modifications generate an active fluorescent intein that can be fused to a target gene
- NPT II beta-gal
- the invention provides methods for modifying inteins, particularly intein splicing regions, e.g., intein splicing regions in the chimeric compositions of the invention.
- Critical regions for intein splicing have been extensively characterized, see, e.g., Chong (1997) J. of Biol. Chem. 272:15587-15590; Telenti (1997) J. of Bacteriol. 179:6378-6382; Chong (1996) J. of Biol. Chem. 271:22159-22168; Derbyshire (1997) Proc. Natl. Acad. Sci. USA 94:11466-11471; Perler (1997) Nucleic Acids Res. 25: 1087-1093.
- an intein used in the compositions and/or methods of the invention comprises only intein sequence(s) critical for splicing.
- an advantage of an intein-detectable moiety (e.g., FP) chimeric construct of the invention over a traditional gene fusion to a detectable moiety (e.g., FP) is that the resulting splicing that liberates the protein of interest (encoded by a gene of interest, GOI) from the intein-detectable moiety (e.g., FP) reporter section of the chimeric composition.
- a gene of interest e.g., FP
- This can be important because in many cases the fusion of even a small affinity tag to either end of a gene or polypeptide can significantly alter gene and/or polypeptide expression, nucleic acid and/or protein stability and/or activity.
- intein constructs with good splicing efficiencies are provided.
- the invention also provides methods for making and screening for intein constructs with good splicing efficiencies.
- the invention provides methods for intein modification comprising evolving conditional mutants to effect in vitro splicing for protein purification, see, e.g., Telenti (1997) J. of Bacteriol. 179:6378-6382; Wood (1999) Nature Biotechnology 17: 889-892; Southworth (2000) EMBO Journal 19:5019-5026.
- intein modification comprises in vivo or in vitro splicing behavior. Mutations that decreased or increased in vivo activity were screened.
- the invention provides methods for determining important intein domains for catalytic activity or other modification of intein behavior.
- inteins are evolved to have temperature, pH and DTT dependence and/or in vitro and/or in vivo cleavage activity, see, e.g., Perler (2000) Nucleic Acids Res. 28:344-345.
- the pEKIl (pEKintl) construct will only provide kanamycin resistance if the intein splices. Many of the mutations generated by GSSM or other techniques will most likely inactivate intein activity.
- a large library of intein mutants is screened for clones able to splice the intein and, subsequently, grow in the presence of kanamycin, see Figure 10.
- positive clones are characterized in a quantitative assay for kanamycin activity to determine which clones splice more efficiently.
- the invention provides a method comprising a combination of a powerful antibiotic selection and a quantitative assay. This can enable the rapid evaluation of large numbers of intein mutants to evolve a more efficiently splicing intein.
- the intein used in the methods and the compositions of the invention have a splicing efficiency of over about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more.
- the amount of unspliced fusion protein e.g., KanR:Intein-FP
- the amount of unspliced fusion protein is less than about 1%, 5%, 10%, 15%, 20% or more of the spliced protein (e.g., KanR).
- estimation of protein levels is by SDS-PAGE and/or by mass spectrometer (MS). MS can quickly and accurately determine the exact size and amount of proteins present in a sample. If large numbers of samples require quantitation of splicing efficiency, mass spectrometry can be used.
- the intein of the invention and the inteins used in the chimeric compositions and polypeptides of the invention have only splicing or only cleaving activity (including substantially splicing or substantially cleaving activity). In one aspect, the intein of the invention and the inteins used in the chimeric compositions and polypeptides of the invention exhibit cleavage rather than splicing when fused to the C-terminus of a target gene. In one aspect, the intein of the invention exhibits cleavage rather than splicing when fused to the C-terminus of a target gene. This cleavage behavior may be used to broaden the applicability of the intein vectors of the invention.
- the intein is fused between the target gene as an "N-extein” and a His6 affinity tag as a "C-extein.”
- the target gene is rejoined to the affinity tag. If cleavage rather than splicing occurs, the target gene is liberated with its native C-terminus and the affinity tag remains fused to the intein. In many cases, it may be necessary to retain the native C-terminus of the target gene for proper protein function.
- the pEKI2 (pEKInt2) construct (see Fig. 2) will only confer kanamycin resistance if the intein cleaves rather than splices.
- pEKI2 pEKInt2 construct the intein cleaves.
- pEKI2 (pEKInt2) construct is used to select for intein mutants that undergo significant cleavage.
- gene evolution technologies are combined with the selection screening for intein cleavage to generate intein constructs that undergo efficient cleavage rather than splicing.
- greater than about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%), 99%, or more of the expressed protein is cleaved as assayed by protein gel and/or mass spectrometry.
- the intein of the invention and the inteins used in the chimeric compositions and polypeptides of the invention function efficiently regardless of the target gene to which they are joined. Also, in one aspect, the intein of the invention and the inteins used in the chimeric compositions and polypeptides of the invention function generate greater than about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more splicing and/or cleavage with a variety of target genes.
- intein activity may adversely affect intein activity.
- the amino acid residue located at the N-terminus of an intein i.e., at the -1 position
- intein constructs with different target genes are screened with a wide range of amino acids at the -1 position.
- the intein is evolved to decrease its sensitivity to the sequence located at the C-terminus of the intein.
- a variety of apparatus and methodologies can be used to in conjunction with the polypeptides and nucleic acids of the invention, e.g., to screen polypeptides for intein activity, to screen compounds as potential modulators, e.g., activators or inhibitors, of an intein activity, for antibodies that bind to a polypeptide of the invention, for nucleic acids that hybridize to a nucleic acid of the invention, to screen for cells expressing a polypeptide of the invention and the like.
- invention provides activity screens using the chimeric compositions of the invention to normalize gene expression, wherein the chimeric compositions of the invention can splice out (cleave out) an enzyme domain or a binding protein domain with about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more efficiency with a variety of target genes.
- Capillary a ⁇ ays such as the GIGAMATRIXTM, Diversa Corporation, 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 a ⁇ ay, including capillary a ⁇ ays.
- a ⁇ ays 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 a ⁇ ays provide another system for holding and screening samples.
- a sample screening apparatus can include a plurality of capillaries formed into an a ⁇ ay 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 a ⁇ ay, 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 a ⁇ ay 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 a ⁇ ay.
- Each capillary of the capillary a ⁇ ay 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 a ⁇ ay, wherein each capillary of the capillary a ⁇ ay 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 a ⁇ ay 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 a ⁇ ay 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 a ⁇ ay can be formed of any number of individual capillaries, for example, a range from 100 to 4,000,000 capillaries.
- a capillary a ⁇ ay can form a micro titer plate having about 100,000 or more individual capillaries bound together.
- Nucleic acids or polypeptides of the invention can be immobilized to or applied to an a ⁇ ay.
- a ⁇ ays 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 intein 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 a ⁇ ay, or "biochip.”
- a ⁇ ay of nucleic acids on a microchip
- some or all of the transcripts of a cell can be simultaneously quantified.
- a ⁇ ays 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 a ⁇ ays can also be used to simultaneously quantify a plurality of proteins.
- the present invention can be practiced with any known “a ⁇ ay,” also refe ⁇ ed to as a “microa ⁇ ay” or “nucleic acid a ⁇ ay” or “polypeptide a ⁇ ay” or “antibody a ⁇ ay” or “biochip,” or variation thereof.
- a ⁇ ays 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.
- target elements 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.
- any known a ⁇ ay and/or method of making and using a ⁇ ays 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;
- compositions and methods of the invention are used in quantitative screens to normalize gene expression.
- the compositions and methods of the invention can be used with any quantitative screen.
- a relatively simple step is added to assay the fluorescence of the chimeric compositions of the invention (e.g., the intein-FP).
- any screening platform can be used.
- the methods of the invention can include microtiter plates, e.g., with 96, 384, or 1536 wells each.
- clones expressing ⁇ -galactosidase activity are detected by adding the chromogenic substrate 2-nitrophenyl ⁇ -D-galactopyranoside (ONPG) to bacteria grown in microtiter plates.
- the invention uses sensitive, cell-permeable, fluorogenic substrates like ⁇ -D-galactopyranoside (e.g., by Molecular Probes #R-1159).
- the cells can be grown in the presence of the substrate.
- the invention provides enzyme activity assays involving multiple steps, e.g., multiple liquid transfer steps, cell lysis step(s), and characterization(s) of reaction products by HPLC, MS or other means.
- the methods of the invention are practiced in wells, e.g., microtiter plate wells.
- smaller well volumes are used to increase throughput and decrease reagent costs.
- smaller well volumes may also increase variability due to growth, expression, and liquid transfer differences.
- GigaMatrixTM is used in the methods of the invention.
- GigaMatrixTM is used for fluorescent detection from samples in very small wells, for example, as small as 250 nL.
- a GigaMatrixTM plate can contain 100,000 wells in the same footprint as a standard microtiter plate. This screening format may offer advantages in screening throughput and reagent use.
- the GigaMatrixTM system has been used to screen libraries for a number of enzyme classes using different fluorogenic substrates.
- the invention provides very high throughput assays using, e.g., a Fluorescence Activated Cell Sorter (FACS) to assay enzyme activity in a single cell format.
- FACS Fluorescence Activated Cell Sorter
- a FACS instrument measures multiple fluorescent signals from single cells at a rate greater than 10 7 cells/min.
- the invention uses a substrate that is cell-permeable, non-toxic and/or non-fluorescent.
- the substrate becomes fluorescent and remains inside the cell instead of diffusing out.
- Several classes of enzymes can be used in the chimeric compositions of the invention and used in methods of the invention comprising a FACS assay (e.g., to assay enzyme activity in a single cell format).
- any assay can be used in the methods of the invention for gene expression normalization.
- invention provides assays with sample sizes decreasing from millions of cells in a microtiter plate to a single cell in a FACS assay. As the sample sizes decrease, the variations in growth and expression become more significant. In a larger sample size, these variations average out in a larger population of cells. Normalization using the compositions and methods of the invention can co ⁇ ect for variations and enable accurate, quantitative measurements of enzyme activity.
- the normalized assays of the invention and/or compositions of the invention are used to develop enzymes for the production of high value chiral compounds. These assays can use, e.g., racemases, nitrilases and epoxide hydrolases. In one aspect, the normalized assays of the invention and/or compositions of the invention are used to develop enzymes for the biocatalytic production of high value pharmaceutical compounds.
- the normalized assays of the invention and/or compositions of the invention are used to develop enzymes, e.g., racemases, hydrolases and nitrilases, that exhibit a variety of different properties and activity profiles, including varied expression, stability, pH and temperature optimums, substrate specificity and enantioselectivity.
- the normalized assays of the invention and/or compositions of the invention are used in the enantioselective production of high value amino acids. High throughput spectroscopic methods can be used. The exemplary method described in Figure 12 can be used to quantitate the enantioselective conversion of amino acids using secondary detection enzymes.
- Figure 12 illustrates an exemplary method for the quantitation of enantioselective nitrilase conversion.
- the D-amino acid oxidase (D-AAO) and L-amino acid oxidase (L-AAO) reactions can be set up in parallel on microtiter plates, such that one is subjected to treatment with the L-processing enzyme and the other is treated with the D-processing enzyme.
- the amount of L-amino acid generated is then detected by converting it to the alpha-keto acid. This oxidation is accompanied by a concomitant release of ammonia and hydrogen peroxide.
- the H 2 O 2 generated is then detected, e.g., using a highly specific and sensitive assay, such as using horseradish peroxidase conducted in the presence of a chromogenic hydrogen donor, such as phenol:4-amino-antipyrine whose oxidation yields a red chromophore.
- a highly specific and sensitive assay such as using horseradish peroxidase conducted in the presence of a chromogenic hydrogen donor, such as phenol:4-amino-antipyrine whose oxidation yields a red chromophore.
- Enantioselectivity can be calculated by comparison of the parallel assays.
- the enantioselectivity of the nitrilase reaction will be assayed in parallel using a D- specific or L-specific amino acid oxidase.
- Preliminary research has shown that levels of D- and L- amino acids can be measured by splitting the nitrilase reaction mixture into two and assaying using the
- This assay can be used to quantitatively screen libraries of enzyme mutants, e.g., those generated by GSSM and GeneReassembly technologies. Screening libraries for increased enantioselectivity using this method may not be sensitive to differences in growth and enzyme expression because the calculation is based on the ratio of L- to D- amino acids and is independent of the total amount of enzyme conversion. However, normalizing activity levels using the intein-FP vectors may allow mutants to be selected based on both their enantioselectivity and their specific activity.
- measuring the intein-FP fluorescence in the secondary reactions may co ⁇ ect for liquid transfer e ⁇ ors introduced when the original nitrilase reaction is split into two.
- the invention provides a single step assay using the normalization methods and compositions of the invention.
- the nitrilase reaction and the secondary amino acid oxidase reaction are performed concu ⁇ ently. This can eliminate the need to split the nitrilase reaction into two separate assays.
- the parallel assays can be performed on duplicate samples grown separately. By eliminating the liquid transfer steps, this can reduce the time and effort of this assay and enable the reduction of reaction size to 1536 well plates or smaller.
- compositions of the invention e.g., a chimeric intein-FP
- normalization methods of the invention can reduce screening efforts and cost, increase sensitivity and productivity, and enable evolution of more effective nitrilases for the production of high value chiral amino acids.
- assays are adapted to capillary a ⁇ ay systems, e.g., the GigaMatrixTM screening platforms.
- GigaMatrixTM wells can be filled with growth media containing an aminonitrile substrate, amino acid oxidase secondary enzyme and a fluorescent hydrogen donor to detect H 2 O 2 production.
- E. coli harboring the library of nitrilase mutants to be screened can be diluted such that an average of one cell per well will be added to each well.
- the production of a D- or L- amino acid can generate a fluorescent signal due to the coupled reaction with the amino acid oxidase.
- the production of both the L- and D- amino acids is directly measured.
- the methods of the invention provide a very rapid primary screen to detect mutants that produce a certain enantiomer. Positive clones can be recovered from the wells and characterized in more detail using a secondary assay. Variations in growth and expression can be even more dramatic in small volumes of the GigaMatrixTM wells compared to the larger well of a microtiter plate.
- the addition of gene expression normalization methods of the invention greatly enhances the sensitivity and quantitative of these assays.
- the bacterial clones grow in the reaction media.
- use of GigaMatrixTM varies depending on a number of factors, including substrate/product, permeability/toxicity and fluorescence sensitivity.
- Figure 13 describes an exemplary nitrilase activity assay using the fluorogenic reagent dihydroxyphenoxazine. This method has been successfully used with several different aminonitriles, demonstrating that these substrates are sufficiently non-toxic and cell permeable to allow sensitive detection.
- the chimeric compositions and methods of the invention are used in enzyme assays, e.g., racemase, nitrilase or epoxide hydroiase assays, for the production of chiral compositions, e.g., epoxides and diols, e.g., for use as pharmaceutical intermediates.
- the methods comprise spectroscopic and fluorescence based measurements of enzyme activities, e.g., epoxide hydroiase activity.
- These assays can utilize synthesized substrate analogs to generate a chromogenic or fluorescent product, as illustrated in Figure 14.
- Figure 14 schematically illustrates exemplary epoxide hydroiase screens.
- Figure 14A illustrates an exemplary fluorogenic epoxide assays using a fluorogenic epoxide substrate. Epoxide hydroiase activity can generate a fluorescent signal when this synthetic substrate is cleaved.
- Figure B illustrates an exemplary chromogenic epoxide assay. Epoxide hydroiase activity can reduce the color change when this assay is performed.
- chromogenic and fluorogenic epoxide hydroiase substrates were synthesized at Diversa. Using these substrates in the assay, the activity of a putative epoxide hydroiase was verified. As in the nitrilase assay, the fluorescent detection of epoxide hydroiase activity could be adapted to high density microtiter plates or the GigaMatrix format. Using the intein-FP technologies, these assays will be more quantitative, enabling the development of additional epoxide hydroiase enzymes that will produce specific high value compounds.
- activity in the methods of the invention is measured by a variety of biological and analytical methods. Any assay providing a quantitative value for activity can be normalized to protein expression using the methods of the invention.
- Intein-detectable moiety e.g., intein-FP fluorescence can be measured in a variety of formats.
- the normalized assays including both the nitrilase and epoxide hydroiase assays of the invention, are used in high throughput screening.
- the methods of the invention provide increased data quality and sensitivity and lower background "noise" from variations in growth, expression, and pipetting.
- the additional information provided by the normalization methods of the invention is used to compare enzymes based on their specific activity rather than whole cell activity. Screening libraries for modified proteins using intein normalized data
- the invention provides methods for screening libraries for modified enzymes or binding proteins (e.g., improved or other altered activity) using intein normalized data generated by the methods of the invention.
- a specific target for this project is nitrilases, e.g., those for the production of intermediates for ACE inhibitors.
- the goal is to evolve an appropriate enzyme to produce key intermediates, e.g., 4-phenyl-2-amino-butanoic acid, for manufacturing these drugs.
- the invention also provides methods for developing a process for using this enzyme to manufacture the 4-phenyl-2-amino-butanoic acid intermediate. This includes developing a production process for 4-phenyl-2-amino-butanoic acid.
- the processes include various enzyme properties, including variations in enantioselectivity, specific activity, stability, expression, etc.
- the methods of the invention can incorporate any nucleic acid or polypeptide modification schemes, including GSSM and or GeneReassembly technologies described above.
- mutant libraries are constructed and screened for the desired enzyme properties. Hits from this screening effort can be characterized in more detail and can serve as a template for a second round of evolution to further modify the enzyme's characteristics. This iterative cycle of mutagenesis and screening can be repeated until a desired protein, e.g., enzyme or binding protein, has been developed.
- the intein technology of the invention provides an efficient means to express genes in a one-to-one ratio with a detectable label, e.g., a fluorescent protein.
- a detectable label e.g., a fluorescent protein.
- the methods of the invention can increase the sensitivity and productivity of various screening platforms and enable the development of novel screening methods.
- the methods of the invention for normalizing gene expression in living cells have many applications in academic and industrial areas.
- the invention provides novel screens for the development of enzymes for the production of high value chiral molecules. These enzymes can catalyze the selective
- the enzymes generated by this technology can be used in production processes to meet demand for chiral molecules discussed in this document.
- the methods of the invention can be used to discover, develop, manufacture and employ enzymes for the production of chiral intermediates and active pharmaceutical ingredients for pharmaceuticals, such as chiral building blocks, enzyme libraries and bulk enzymes.
- the methods of the invention can be used for nitrilase and epoxide hydroiase biotransformation.
- the exemplary enzyme targets for use with the methods of the invention are set forth in the following table:
- the chimeric compositions of the invention can comprise any known intein, of which there is a wide range of sequence diversity, splicing behavior, and structures. These sequences can be modified by any technique or methodology, e.g., using gene reassembly techniques, e.g., GeneReassemblyTM, to recombine elements of the different inteins to generate diverse libraries.
- the invention provides a method comprising GeneReassemblyTM to recombine domains from a family of genes. The method of the invention combines domains from different inteins to create inteins and chimeric proteins with novel properties.
- FIG. 8 An exemplary method of the invention for making libraries of inteins using GeneReassemblyTM is set forth in Figure 8.
- these libraries are screened for mutants with different (e.g., increased) splicing and/or cleavage efficiency.
- the libraries can be cloned into an cloning vehicle, e.g., a vector, to facilitate the screening and/or to recombine elements of the different inteins.
- an antibiotic immunoactivity assay can be used, e.g., a Kanamycin immunoactivity assay as described, e.g., in Henderson (1991) Analytical Biochem. 194:64-68.
- this assay involves immunoaffinity immobilization of an NPTII protein (the protein conferring kanamycin resistance, also called KanR) in the sample.
- NPTII protein the protein conferring kanamycin resistance, also called KanR
- it is followed by a secondary enzyme reaction to measure NPTII activity.
- the invention provides methods comprising the combination of a selection screen and a high throughput activity screen.
- an exemplary methods comprise a growth selection for nitrilase activity and a quantitative high throughput nitrilase assay.
- a nitrilase is used a construct for the selection and activity screen.
- a mutant library is selected in a bacterial host, e.g., an E. coli host, that requires an active nitrilase to grow on a minimal medium supplemented with a nitrile as the sole nitrogen source. Only clones containing an active intein will have nitrilase activity and thus the ability to grow on this medium.
- these selected clones can be then be assayed in a high throughput activity screen to measure nitrilase activity using a fluorescent substrate as an activity indicator.
- this screen has been used to screen up to 20,000 clones or more per day and detect increases in activity as low as 10%.
- the invention provides isolated or recombinant antibodies that specifically bind to an intein of the invention. These antibodies can be used to isolate, identify or quantify the inteins of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention or other related inteins. The antibodies can be designed to bind to a splice or cleavage site of an intein.
- 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 a ⁇ ay 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, e.g., the inteins, 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.
- 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. For preparation of monoclonal antibodies, 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).
- Techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies to the polypeptides of the invention.
- transgenic mice may be used to express humanized antibodies to these polypeptides or fragments thereof.
- Antibodies generated against the polypeptides of the invention may be used in screening for similar polypeptides (e.g., inteins) from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody and those polypeptides which specifically bind the antibody are detected. Any of the procedures described above may be used to. detect antibody binding.
- polypeptides e.g., inteins
- Kits The invention provides kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, transgenic seeds or plants or plant parts, polypeptides (e.g., inteins) 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.
- the methods of the invention for normalizing gene expression in assays can be used in whole cell evolution, or whole cell engineering, of a cell to develop a new cell strain having a new phenotype, e.g., a new or modified enzyme activity, a modified intein activity.
- the genetic composition of the cell is modified.
- the genetic composition can be modified by addition to the cell of a nucleic acid of the invention.
- To detect the new phenotype 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 inteins 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 confe ⁇ ed and detected, including new or improved characteristics in the cell. Any aspect of metabolism or growth can be monitored.
- the invention provides a chimeric protein comprising at least three domains, wherein one domain comprises a detectable moiety domain.
- the detectable moiety can be any detectable composition, including fluorescent, bioluminescent, chemiluminescent or radioactive moieties.
- Bioluminescence includes all bioluminescence, fluorescence or chemiluminescence or other photon detectable systems.
- Bioluminescent and chemiluminescent polypeptides used in the compositions and methods of the invention include all known polypeptides known to be bioluminescent or chemiluminescent, or, acting as enzymes on a specific substrate (reagent), can generate (by their enzymatic action) a bioluminescent or chemiluminescent molecule. They include, e.g., isolated and recombinant luciferases, aequorin, obelin, mnemiopsin, berovin and variations thereof and combinations thereof, as discussed in detail, below.
- the bioluminescent or chemiluminescent are enzymes that act on a substrate that reacts with the reagent in situ to generate a molecule that can be imaged.
- the substrate can be administered before, at the same time (e.g., in the same formulation), or after administration of the chimeric polypeptide (including the enzyme).
- these polypeptides include, e.g., luciferase, aequorin, halistaurin, phialidin, obelin, mnemiopsin or berovin, or, equivalent photoproteins, and combinations thereof.
- the compositions and methods of the invention also include recombinant forms of these polypeptides as recombinant chimeric or "fusion" proteins, including chimeric nucleic acids and constructs encoding them. Methods of making recombinant forms of these polypeptides are well known in the art, e.g., luciferase reporter plasmids are described, e.g., by Everett (1999) J. Steroid Biochem. Mol.
- the monomeric Ca2+-binding protein aequorin is described by, e.g., Kurose (1989) Proc. Natl. Acad. Sci. USA 86:80-84; Shimomura (1995) Biochem. Biophys. Res. Commun. 211 :359-363.
- the aequorin-type photoproteins halistaurin and phialidin are described by, e.g., Shimomura (1985) Biochem J. 228:745-749. Ward (1975) Proc. Natl. Acad. Sci
- bioluminescent or chemiluminescent chimeric polypeptides of the invention can be made by any method, see, e.g., U.S. Patent No. 6,087,476, that describes making recombinant, chimeric luminescent proteins.
- U.S. Patent Nos. 6,143,50; 6,074,859; 6,074,859, 5,229,285, describe making recombinant luminescent proteins.
- bioluminescent or chemiluminescent activity of the chimeric recombinant polypeptides of the invention can be assayed, e.g., using assays described in, e.g., U.S. Patent Nos. 6,132,983; 6,087,476; 6,060,261; 5,866,348; 5,094,939; 5,744,320.
- Various photoproteins that can be used in compositions of the invention are described in, e.g., U.S. Patent Nos. 5,648,218; 5,360,728; 5,098,828.
- compositions and methods of the invention include use of any device capable of detecting bioluminescence, fluorescence or chemiluminescence or other photon detection systems.
- the methods of the invention can be practiced using any photon detection device, or variation or equivalent thereof, or in conjunction with any known photon detection methodology, including visual imaging.
- An exemplary photodetector device is an intensified charge-coupled device (ICCD) camera coupled to an image processor. See, e.g., U.S. Patent No. 5,650,135.
- Photon detection devices are manufactured by, e.g., Xenogen (Alameda, CA) (the Xenogen IVISTM imaging system); or, Hamamatsu Corp., Bridgewater, NJ.
- Another imaging system used for compositions and methods of the invention can be a proximal charge-coupled device (CCD) detection/imaging. Due to its inherent versatility, it can also accommodate chemiluminescence, fluorescent and radioisotope target molecule detection, high throughput, and high sensitivity.
- CCD charge-coupled device
- This detection/imaging apparatus can include a lensless imaging a ⁇ ay comprising a plurality of solid state imaging devices, such as an a ⁇ ay of CCDs, photoconductor-on-MOS a ⁇ ays, photoconductor-on-CMOS a ⁇ ays, charge injection devices (CIDs), photoconductor on thin-film transistor a ⁇ ays, amo ⁇ hous silicon sensors, photodiode a ⁇ ays, or the like.
- CCDs charge injection devices
- CDs charge injection devices
- the compositions and methods of the invention inco ⁇ orate in whole or in part designs of detection devices as described, e.g., in U.S. Patent Nos.
- compositions and methods of the invention inco ⁇ orate and use any detectable label.
- exemplary labels include, e.g., 32 P, 35 S, 3 H, 14 C, 125 1, 131 I; fluorescent dyes (e.g., Cy5TM, Cy3TM, FITC, rhodamine, lanthanide phosphors, Texas red), electron- dense reagents (e.g. gold), enzymes, e.g., as commonly used in an ELISA (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g. colloidal gold), magnetic labels (e.g.
- DynabeadsTM biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
- the label can be directly inco ⁇ orated into the nucleic acid or other target compound to be detected, or it can be attached to a probe or antibody that hybridizes or binds to the target.
- a peptide can be made detectable by inco ⁇ orating (e.g., into a nucleoside base) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, transcriptional activator polypeptide, metal binding domains, epitope tags).
- Label can be attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties (the domains of the chimeric compositions can also be separated by various spacers). See, e.g., Mansfield (1995) Mol Cell Probes 9:145-156. Cyanine and related dyes, such as merocyanine, styryl and oxonol dyes, are particularly strongly light-absorbing and highly luminescent, see, e.g., U.S. Patent Nos. 4,337,063; 4,404,289; 6,048,982. Cy3TM and Cy5TM can be used together; both are fluorescent cyanine dyes produced by Amersham Life Sciences (Arlington Heights, IL).
- labeling with a detectable composition also can include a nucleic acid attached to another biological molecule, such as a nucleic acid, e.g., a nucleic acid in the form of a stem-loop structure as a "molecular beacon” or an "aptamer beacon.”
- a nucleic acid e.g., a nucleic acid in the form of a stem-loop structure as a "molecular beacon” or an "aptamer beacon.”
- Molecular beacons as detectable moieties are well known in the art; for example, Sokol (1998) Proc. Natl. Acad. Sci. USA 95:11538-11543, synthesized "molecular beacon” reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends.
- the molecular beacon In the absence of a complementary nucleic acid strand, the molecular beacon remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission.
- the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength.
- Antony 2001
- Biochemistry 40:9387- 9395 describing a molecular beacon comprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. See also U.S. Patent Nos. 6,277,581 and 6,235,504.
- Aptamer beacons are similar to molecular beacons; see, e.g., Hamaguchi (2001) Anal. Biochem. 294:126-131; Poddar (2001) Mol. Cell. Probes 15:161-167; Kaboev (2000) Nucleic Acids Res. 28:E94. Aptamer beacons can adopt two or more conformations, one of which allows ligand binding. A fluorescence-quenching pair is used to report changes in conformation induced by ligand binding. See also, e.g., Yamamoto (2000) Genes Cells 5:389-396; Smirnov (2000) Biochemistry 39:1462-1468.
- the vector can comprise a fluorescent protein (FP) inserted into an intein and fused downstream of a gene of interest (GOI), see Figure 1, a schematic describing the strategy for developing an exemplary intein expression vector.
- FP fluorescent protein
- GOI gene of interest
- inteins from the vacuolar ATPase subunit of Saccharomyces cerevisiae See VMA
- GFP Green Fluorescent Protein
- nrdF ribonucleoside diphosphate reductase
- the intein from the ribonucleoside diphosphate reductase (nrdF) gene of the Aquifex aeolicus genome was determined by sequence homology to contain an intein encoding 346 amino acid residues. Its ability to catalyze its own excision from its native protein precursor and from within the context of a foreign protein was not previously demonstrated.
- FP's from Clontech in a variety of different colors were also used; they behave in a similar manner to GFP in terms of their bacterial expression and tolerance of N- or C-terminal fusions.
- the neomycin phosphotransferase gene (NPT II) (hereafter denoted as KanR) provides kanamycin resistance and was used as a marker for preliminary screening of the intein fusions.
- the entire intein-containing nrdF gene was PCR amplified from Aquifex aeolicus (SEQ ID NO: 1) and overexpressed in E. coli. Protein gel analysis clearly showed efficient splicing of the Aquifex intein from its native gene, confirming its intein activity.
- inteins When most inteins splice from their natural protein precursor, they leave behind a cysteine at the site of insertion. Thus, it is possible to insert the intein sequence at any cysteine in a target gene. Constructs were made with the Aquifex intein cloned into either cysteine33 or at the C-terminus of the KanR gene and subcloned into the pET21b (Novagen) vector behind the T7 promoter to generate plasmids pEKIl (pEKintl) and pEKI2 (pEKint2), respectively; see Figure 2, a diagram of the constructs used to generate exemplary intein expression vector.
- the KanR gene does not normally have a cysteine at its C-terminus, so one was added for the construction of pEKI2.
- the KanR:Intein fusions were also subcloned as fusions to a hexahistidine (His 6 ) tag for affinity purification and Western detection of the spliced products.
- KanR was subcloned in the same vector with its native C-terminus, an additional cysteine at the C-terminus or a His 6 affinity tag to generate pEK, pEKCys, and pEKH respectively.
- E. coli carrying each plasmid was tested for growth in the presence of kanamycin at 25 ⁇ g/ml (see Fig. 2).
- the pEKIl (pEKintl) plasmid was able to confer kanamycin resistance, confirming the splicing activity of the Aquifex intein in a foreign context.
- the pEKI2H (pEKInt2H) plasmid was also able to confer kanamycin resistance. Combined with the fact that a His 6 tag destroys KanR function, this strongly suggested that the kanamycin resistance of the pEKI2H was a result of a cleavage event at the N-terminus of the intein instead of splicing.
- K kanamycin
- KH kanamycin with His6 tag
- KI1H kanamycin with intein and His6 tag
- II spliced intein
- KI2H kanamycin with intein and His6 tag
- I2H intein with His6 tag.
- KanRH His6 tagged KanR protein
- SEQ ID NO: 1 encoding SEQ ID NO: 2.
- a 30 kDa was visible, which likely co ⁇ esponds to KanRH. This strongly suggests that the intein spliced and KanR containing the His6 tag was regenerated.
- a 29 kDa protein co ⁇ esponding to KanR was detected in the pEKI2H sample providing solid evidence that the intein cleaved at its N-terminus. In this case, the His6 tag remained fused to the intein and KanR was unaltered.
- FP fluorescent proteins
- flexible linkers are added to the chimeric polypeptides of the invention.
- the detectable moiety e.g., fluorescent protein
- Figure 6A Flexible linkers are introduced between the intein and FP domains to relax steric interference between the domains.
- Figure B Fusion of the GFP C-terminal to the intein. After translation, the intein will cleave, releasing the GOI without any C-terminal additions.
- the Green and Cyan FP's from Clontech were inserted into the VMA intein at residue 204 or replacing residues 204-383. These residues were shown to be dispensable for intein activity if replaced by a small flexible linker; see, e.g., Chong (1997) J. of Biol. Chem. 272:15587-15590.
- the lacZ gene fromE. coli and two other ⁇ - galactosidase genes were fused in place of KanR to the N-terminus of the VMA and VMA-FP constructs. These ⁇ -gal genes were chosen because they represent a range of specific activities and expression levels. When overexpressed in E.
- the exemplary Aquifex intein chosen for this project proved to be difficult to express and had significantly, reduced splicing activity when inserted into a foreign context.
- constructs were made by inserting the different FP genes into the Aquifex or VMA inteins, neither intein activity nor FP fluorescence were observed.
- Manipulation of the VMA and other inteins has shown that a flexible linker between the N- and C- terminal intein motifs is required (Chong (1997) J. of Biol. Chem. 272:15587-15590).
- the configurations in which the FP's were inserted may have caused structural interference leading to a nonfunctional intein. This interference may also explain the lack of FP fluorescence.
- the addition of flexible linkers between the intein and FP sequences may relax the conformation allowing the intein and FP domains to function normally.
- the FP is fused to the C-terminus of the intein, as illustrated in Figure 6.
- the cleavage activity of the Aquifex intein produces the desired protein.
- the invention provides chimeric compositions (e.g., fusion proteins) comprising linker insertions between their domains.
- the intein domains may require proper folding for efficient splicing or cleavage to occur.
- the reacting groups may have to align precisely.
- Chong 1997 J. of Biol. Chem. 272:15587-15590
- a portion of the intein encoding an endonuclease was deleted.
- the resulting mini-intein failed to exhibit splicing.
- two flexible peptide linkers were inserted into the site that was deleted, which resulted in efficient splicing in vivo.
- linker insertion to improve the splicing of a mini-intein was demonstrated in the Mxe GyrA intein from Mycobacterium xenopi, see, e.g., Telenti (1997) J. of Bacteriol. 179:6378- 6382. Any linker, including those described by Chong (1997) or Telenti (1997), can be used in chimeric compositions of the invention. Routine screening can be done on known inteins to generate optimal mini-inteins for particular pu ⁇ oses.
- chimeric compositions of the invention further comprise a domain for affinity purification, such as a chitin binding domain, a His6 tag, any epitope for Ab affinity ligation, and the like.
- the chimeric compositions of the invention can be a mini-intein vector comprising a domain for affinity purification.
- the polypeptide can be a modified intein fused to a target protein at its N-terminus and a chitin binding domain-GFP fusion at its C-terminus, as described by Zhang (2001) Gene 275:241-252. This permits monitoring of soluble protein expression by following GFP fluorescence combined with an option for affinity purification. In Zhang (2001), cleavage was only observed in vitro.
- Zhang (2001) an intein fused in frame to GFP resulted in a functional intein albeit in vitro and a functional fluorescence.
- cleavage e.g., from a chimeric composition as illustrated in Figure 6B
- Exemplary chimeric compositions comprising inteins can undergo cleavage instead of splicing and can be used in normalization technology.
- inteins are used that enable the expression of gene products having their native C-termini.
- a small affinity tag is ligated at the C-terminus.
- antibiotic selections are used for both splicing and cleavage events. These selections can be used to develop additional, including more efficient, inteins in Phase II.
- a pH sensitive GFP is inco ⁇ orated into a chimeric composition of the invention. It can be used to detect proton production by an enzyme, e.g., a hydrolytic enzyme.
- a chimeric composition comprises a pH sensitive GFP and a hydrolytic enzyme (and an intein).
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WO2005005654A1 (en) * | 2003-07-11 | 2005-01-20 | Novozymes A/S | Method of screening for improved specific activity of enzymes |
US8420387B2 (en) * | 2009-11-06 | 2013-04-16 | Agrivida, Inc. | Intein-modified enzymes, their production and industrial applications |
US20130071884A1 (en) | 2009-11-06 | 2013-03-21 | Agrivida, Inc. | Plants expressing cell wall degrading enzymes and expression vectors |
US10407742B2 (en) | 2009-11-06 | 2019-09-10 | Agrivida, Inc. | Intein-modified enzymes, their production and industrial applications |
US9464333B2 (en) | 2009-11-06 | 2016-10-11 | Agrivida, Inc. | Intein-modified enzymes, their production and industrial applications |
US10443068B2 (en) | 2010-06-25 | 2019-10-15 | Agrivida, Inc. | Plants with engineered endogenous genes |
US9598700B2 (en) | 2010-06-25 | 2017-03-21 | Agrivida, Inc. | Methods and compositions for processing biomass with elevated levels of starch |
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