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
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides

Definitions

• the present invention essentially consists of using a coupled enzymatic reaction wherein the end product of the reaction is preferably used as a catalyst in the first step of the reaction. Once the reaction is "primed” with a small amount of product, the reaction will accelerate as more catalyst, i.e. the end product, is made. The result is that the final mixture is not contaminated with any undesired triphosphate such as ATP.
• NTPs Ribonucleoside triphosphates
• dNTPs deoxyribonucleoside triphosphates
• the first step of the nucleotide synthesis involves converting the appropriate ribonucleoside or deoxyribonucleoside monophosphateor derivatives thereof (e.g., xNMP) into the appropriate ribonucleoside or deoxyribonucleoside diphosphate or derivatives thereof (e.g., xNDP) in the presence of a enzyme capable of converting the xNMP into the xNDP, and a phosphate donor.
• xNMP ribonucleoside or deoxyribonucleoside monophosphateor derivatives thereof
• xNDP e.g., xNDP
• the phosphate donor is a catalytic amount of the desired ribonucleotide or deoxyribonucleoside triphosphate or derivatives thereof (e.g., xNTP) to prime the reaction.
• the enzyme may be any kinase and preferably is a base specific nucleoside monophosphate kinase (NMK).
• NMK base specific nucleoside monophosphate kinase
• the enzyme in this step may be any kinase and preferably is a pyruvate kinase (PYK) and the phosphate donor may be any phosphate donor and preferably is phosphoenolpyruvate, PEP.
• the phosphate donors are obtained by an enzymatic reaction. Most preferably, the phosphate donors are synthesized and supplied enzymatically during the nucleotide synthesis process of the invention.
• the phosphate donor PEP is obtained from the reaction of D(-)3-phosphoglyceric acid (3-PGA) in the presence of the enzymes phosphoglycerate mutase (PGM) and enolase (ENO).
• PGM phosphoglycerate mutase
• ENO enolase
• PEP may be generated enzymatically from any other precursor including 2- phosphoglyceric acid (2-PGA).
• the phosphate donor may be made by chemical or other means or obtained commercially. Preferred phosphate donors include PEP, 1,3-diphosphoglycerate, and xNTPs.
• the multiple enzymatic steps are preferably accomplished simultaneously in a reaction chamber or vessel.
• the one or more enzymatic reactions to make the phosphate donors and the one or more enzymatic reactions to make the desired nucleotides are accomplished in a batch process.
• the reaction steps may however also be carried out separately, wherein the products from a previous reaction are added to the next reaction in separate steps.
• the reaction may be carried out as a combination of a batch process and separate steps, where some of the enzymatic steps are accomplished at the same time in one reaction chamber while others are done separately and added to the reaction chamber.
• the present invention relates to a method of making one or more xNMPs comprising (a) mixing one or more nucleosides or derivatives thereof, an enzyme capable of converting said nucleosides into said xNMPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said one or more xNMPs.
• the present invention also relates to a method of making one or more xNDPs comprising
• the present invention further relates to a method of making one or more xNTPs comprising (a) mixing one or more xNDPs, an enzyme capable of converting said xNDPs into xNTPs and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNTPs.
• the phosphate donors are supplied in the reaction by an enzymatic process.
• such methods may further comprise mixing one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the present invention generally relates to a method of making one or more xNDPs comprising (a) mixing one or more nucleosides or derivatives thereof, one or more enzymes capable of converting said nucleosides into xNMPs and converting said xNMPs into xNDPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNDPs.
• the present invention also relates to a method of making one or more xNTPs comprising (a) mixing one or more xNMPs, one or more enzymes capable of converting said xNMPs into xNDPs and converting said xNDPs into xNTPs, and one or more phosphate donors; and
• the present invention further relates to a method of making one or more xNTPs comprising (a) mixing one or more nucleosides or derivatives thereof, one or more enzymes capable of converting said nucleosides into xNMPs, converting said xNMPs into xNDPs, and converting said xNDPs into xNTPs and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNTPs.
• the phosphate donors are supplied in the reaction by an enzymatic process.
• such methods may further comprise mixing one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the present invention also relates to compositions comprising (a) one or more nucleosides or derivatives thereof; (b) at least one enzyme capable of converting said nucleosides into xNMPs; and (c) one or more phosphate donors.
• the present invention also relates to a composition comprising (a) one or more xNMPs; (b) at least one enzyme capable of converting said xNMPs into xNDPs; and (c) one or more phosphate donors.
• the present invention further relates to a composition
• a composition comprising (a) one or more xNDPs; (b) at least one enzyme capable of converting said xNDPs into xNTPs; and (c) one or more phosphate donors.
• Such compositions may further comprise in place of or in addition to the phosphate donors, one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the present invention relates to a composition
• a composition comprising
• the present invention further relates to a composition comprising (a) one or more xNMPs;
• the present invention relates to a composition
• a composition comprising (a) one or more nucleosides or derivatives thereof; (b) one or more enzymes capable of converting said nucleosides into xNMPs, converting said xNMPs into xNDPs, and converting said xNDPs into xNTPs; and (c) one or more phosphate donors.
• Such compositions may further comprise in place of or in addition to the phosphate donors, one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the present invention also concerns a composition
• a composition comprising at least one enzyme capable of converting one or more nucleosides or derivatives thereof into xNMPs and at least one enzyme capable of converting xNMPs into xNDPs; a composition comprising at least one enzyme capable of converting xNMPs into xNDPs and at least one enzyme capable of converting xNDPs into xNTPs; and a composition comprising at least one enzyme capable of converting one or more nucleosides or derivatives thereof into a xNMPs, at least one enzyme capable of converting xNMPs into xNDPs, and at least one enzyme capable of converting xNDPs into xNTPs.
• Such compositions may further comprise one or more phosphate donors, one or more phosphate donor substrates and/or one or more enzymes capable of converting such substrates into phosphate donors.
• kits for the enzymatic preparation for nucleotides comprise at least one component selected from the group consisting of one or more enzymes capable of converting a nucleoside into xNMP, xNMP into xNDP, and/or xNDP into xNTP; one or more enzymes capable of converting one or more phosphate donor substrates into one or more phosphate donors; one or more phosphate donors; one or more phosphate donor substrates; one or more substrates for making the desired nucleotides (e.g., nucleosides, xNMPs, and xNDPs); and instructions or protocols for carrying out the methods of the invention.
• one or more enzymes capable of converting a nucleoside into xNMP, xNMP into xNDP, and/or xNDP into xNTP one or more enzymes capable of converting one or more phosphate donor substrates into one or more phosphate donors; one or more phosphate donors; one or more phosphate donor
• the present invention permits the large scale production of high quality nucleotides which may be used in amplification (PCR), sequencing, labeling and cDNA synthesis.
• PCR amplification
• sequencing labeling
• cDNA synthesis amplification
• the invention relates to nucleotides produced by the methods of the invention and to the use of such nucleotides in standard molecular biology techniques.
• the present invention also relates to the cloning and overexpression of kinases, particularly adenine monophosphate kinase (AMK), guanosine monophosphate kinase (GMK), cytidine monophosphate kinase (CMK), thymidine monophosphate kinase (TMK) and pyruvate kinase (PYK) useful in the invention, as well as the cloning and overexpression of enzymes for synthesizing a phosphate donor, particularly PGM and ENO.
• the expressed enzymes are preferably substantially pure (e.g., >95% pure) which improves the quality of the resulting synthesized product.
• such enzymes are expressed as fusion proteins, preferably having an amino and/or carboxy tag sequence.
• tag sequences are preferably used to assist in isolation and purification of the desired enzyme.
• the invention relates to recombinantly produced enzymes (preferably as fusion proteins) used in the invention, to vectors containing genes encoding such enzymes and to host cells containing such vectors.
• Figure 1 is a flowchart representing synthesis of xNTP according to a preferred embodiment of the present invention.
• FIG. 2 is a flowchart representing the 1,3-diphosphoglycerate enzymatic method to supply phosphate donor according to a preferred embodiment of the present invention.
• Enzymes include gly cerol-3 -phosphate dehydrogenase (1), triose phosphate isomerase (2), glyceraldehyde-3- phosphate dehydrogenase (3), kinases (4 and 5), and lactate dehydrogenase (6).
• Inputs are outlined in bold.
• NAD and xNTP are cofactors that are added in small amounts.
• Figure 3 is a flowchart representing the citric acid enzymatic method to supply phosphate donor according to a preferred embodiment of the present invention.
• the enzymes including aconitase (1), isocitrate dehydrogenase (2), alpha-ketoglutarate dehydrogenase (3), kinases (4 and 5), and lactate dehydrogenase (6). Inputs are outlined in bold. NAD and xNTP are cofactors that are added in small amounts.
• Figure 4 is a flowchart representing the ribulose 1,5-bisphosphate enzymatic method to supply phosphate donor according to a preferred embodiment of the present invention.
• The include ribulose- 1-5-bisphosphate carboxylase (1), PGM (2), ENO (3), and kinases (4 and 5). Inputs are outlined in bold.
• xNTP is a cofactor that is added in small amounts.
• Figure 5 is a flowchart representing the glutamate enzymatic method to supply phosphate donor according to a preferred embodiment of the present invention.
• the enzymes include glutamate dehydrogenase (1), alpha- ketoglutarate dehydrogenase (2), kinase (3), succinyl CoA synthetase (4), and lactate dehydrogenase (5). Inputs are outlined in bold. NAD and xNTP are cofactors that are added in small amounts.
• FIG. 6 is a flowchart representing the use of a single kinase (PKP) for converting xNMP to xNDP and xNDP to xNTP, using PEP as a phosphate donor.
• PEP single kinase
• FIG. 7 is a flowchart representing the use of a single kinase (PKP) for converting nucleoside into xNTP, using PEP as a phosphate donor.
• PEP single kinase
• FIG. 8 is a flowchart representing the use of two kinases (PKP and pyruvate kinase) to convert xNMP to xNTP, using PEP as a phosphate donor.
• PEP two kinases
• Figure 9 is a graph representing the pH profile of dATP synthesis.
• Figure 10 is a graph representing synthesis yield of dGTP and GTP.
• Figure 11 is a graph representing dTTP synthesis on a 3 mmol scale as a function of time as measured by HPLC.
• Figure 12 is a graph representing the effect of equivalents of 3 -PGA on dTTP yield.
• Figure 13 represents HPLC analysis of the reaction product of the enzymatic synthesis of dATP using ATP as a catalyst.
• a representative HPLC chromatogram is depicted, with absorbance plotted as a function of time (in minutes). The time of elution (retention time, in minutes) is displayed above each peak.
• Figure 14 represents HPLC analysis of the purification product of the enzymatic synthesis of dATP using ATP as a catalyst.
• a representative HPLC chromatogram is depicted, with absorbance plotted as a function of time (in minutes). The time of elution (retention time, in minutes) is displayed above each peak.
• Figure 15 represents HPLC analysis of the reaction product of the enzymatic synthesis of dATP using dATP as a catalyst.
• HPLC chromatogram is depicted, with absorbance plotted as a function of time (in minutes). The time of elution (retention time, in minutes) is displayed above each peak.
• Figure 16 represents HPLC analysis of the purification product of the enzymatic synthesis of dATP using dATP as a catalyst.
• a representative HPLC chromatogram is depicted, with absorbance plotted as a function of time (in minutes). The time of elution (retention time, in minutes) is displayed above each peak.
• Figure 17 is a graph representing an assay of CMK by coupling the reaction to NADH oxidation using excess PYK and lactate dehydrogenase as a function of time.
• Figure 18 schematically depicts plasmid pAMP-AMK.
• Figure 19 schematically depicts plasmid pROEX-AMK.
• Figure 20 schematically depicts plasmid pAMP-TMK.
• Figure 21 schematically depicts plasmid pROEX-TMK.
• Figure 22 schematically depicts plasmid pAMP-CMK.
• Figure 23 schematically depicts plasmid pROEX-CMK.
• Figure 24 schematically depicts plasmid pAMP-GMK.
• Figure 25 schematically depicts plasmid pROEX-GMK.
• Figure 26 schematically depicts plasmid pAMP-PGM.
• Figure 27 schematically depicts plasmid pROEX-PGM.
• Figure 28 schematically depicts plasmid pAMP-PYK-CHis.
• Figure 29 schematically depicts plasmid pTrcN-PYK-CHis.
• Figure 30 schematically depicts plasmid pAMP-ENO.
• Figure 31 schematically depicts plasmid pROEX-ENO.
• nucleoside refers to a nitrogen base bound to a sugar molecule.
• the term includes compounds such as adenine, cytidine, guanosine, thymidine, uridine, and AIDS drugs that inhibit reverse transcriptase such as
• the nucleoside may be detectably labeled (e.g. radioactive labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, biotin labels and enzyme labels) or unlabeled.
• nucleotide refers to a base-sugar-phosphate combination, including the corresponding monophosphates, diphosphates and triphosphates. Nucleotides are monomeric units of a nucleic acid sequence
• nucleotide includes ribonucleoside triphosphates such as ATP, CTP, GTP and UTP and derivatives thereof.
• the term further includes deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
• Such derivatives include, for example, [ ⁇ SldATP, 7-deaza-dGTP and 7-deaza-dATP.
• nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
• nucleotide examples include, but are not limited to, ddATP, ddCTP, ddGTP, ddlTP, and ddTTP.
• the term nucleotide also includes the monophosphate and diphosphate corresponding to the above listed triphosphates. Nucleotides and derivatives thereof are generally referred to herein as xNMP for the nucleotide monophosphate and its derivatives, xNDP for the nucleotide diphosphate and its derivatives and xNTP for the nucleotide triphosphate and its derivatives.
• the nucleotide may be unlabeled or detectably labeled. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, biotin labels and enzyme labels.
• xNTP may represent any ribonucleoside triphosphate (NTP) or any deoxyribonucleoside triphosphate (dNTP) and derivatives thereof such as dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof.
• xNMP may represent any ribonucleoside monophosphate (NMP) or any deoxyribonucleoside monophosphate (dNMP) and derivatives thereof
• xNDP may represent any ribonucleoside diphosphate (NDP) or any deoxyribonucleoside diphosphate (dNDP) and derivatives thereof.
• any nucleoside or derivative thereof, xNMP or xNDP may be used as the starting material to make the corresponding desired product.
• the starting material may be ddNMP or ddNDP.
• the starting material may also be the respective nucleoside or derivative thereof.
• Nucleotides with modified bases can be used to study the process of mutagenic DNA replication and repair, which has been shown to be involved in carcinogenesis.
• the methods of the present invention can also be used to synthesize triphosphates of anti-AIDS pharmaceuticals, including AZT, ddl, and ddC.
• nucleotide produced by the present invention may be used in pharmaceuticals and therefore mayt be formulated in a composition comprising one or more nucleotides and a pharmaceutical carrier.
• phosphate donor refers to one or more substrates capable of donating one or more phosphates.
• phosphate donors include but are limited to PEP, a nucleotide (e.g., xNMP, xNDP, xNTP), and 1 ,3-diphosphoglycerate.
• substantially pure means that the desired purified enzyme or nucleotide is essentially free from contaminants.
• the contaminant amount does not substantially affect or does not substantially adversely affect the activity of the enzyme in the desired reaction.
• the contaminant amount does not substantially affect or does not substantially adversely affect a reaction in which the nucleotide is used.
• the amount of contaminating nucleotides (e.g., nucleosides, xNMP and xNDP) in the synthesis of a product (e.g., xNTP) is below 20%, preferably below 15%, more preferably below 10%, more preferably below 5%, still more preferably below
• host cell refers to any prokaryotic or eukaryotic cell that can serve as a source of an enzyme used in the invention and may include recombinant host cells if it is the recipient of a replicable expression vector or cloning vector or desired gene to express the desired enzyme(s).
• Recombinant host cells preferably contain the gene(s) expressing the desired enzyme on a vector but may contain such gene(s) in its chromosome or genome.
• the terms "host” or "host cell” may be used interchangeably herein. For examples of such hosts, see Maniatis et al., "Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982).
• Preferred prokaryotic hosts include, but are not limited to, bacteria of the genus Escherichia (e.g., E. coli), Bacillus, Staphylococcus, Agrobacter (e.g., A. tumefaciens), Streptomyces, Pseudomonas, Salmonella, Serratia, Caryophanon, etc.
• the most preferred prokaryotic host is E. coli.
• Bacterial hosts of particular interest in the present invention include E. coli strains K12, DH10B, DH5 ⁇ and HB101.
• Preferred eukaryotic hosts include, but are not limited to, fungi, fish cells, yeast cells, plant cells and animal cells.
• animal cells are insect cells such as Drosophila cells, Spodoptera Sf9, Sf21 cells and Trichoplusa High- Five cells; nematode cells such as C. elegans cells; and mammalian cells such as COS cells, CHO cells, VERO cells, 293 cells, PERC6 cells, BHK cells and human cells.
• insect cells such as Drosophila cells, Spodoptera Sf9, Sf21 cells and Trichoplusa High- Five cells
• nematode cells such as C. elegans cells
• mammalian cells such as COS cells, CHO cells, VERO cells, 293 cells, PERC6 cells, BHK cells and human cells.
• a vector is a nucleic acid molecule (preferably DNA) capable of replicating autonomously in a host cell.
• Such vectors may also be characterized by having a small number of endonuclease restriction sites at which such sequences may be cut without loss of an essential biological function and into which nucleic acid molecules may be spliced to bring about its replication and expressing. Examples include plasmids, autonomously replicating sequences (ARS), centromeres, cosmids and phagemids.
• Vectors can further provide primer sites, e.g., for PCR, transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, etc.
• the vector can further contain one or more selectable markers suitable for use in the identification of cells transformed or transfected with the vector, such as kanamycin, tetracycline, amplicillin, etc.
• any vector may be used.
• vectors known in the art and those commercially available (and variants or derivatives thereof) may be used in accordance with the invention.
• Such vectors may be obtained from, for example, Vector Laboratories Inc., InVitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, Perkin Elmer, Pharmingen, Life Technologies, Inc., and Research Genetics. Such vectors may be used for cloning or subcloning nucleic acid molecules of interest and therefore recombinant vectors containing inserts, nucleic acid fragments or genes (or portions of genes) may also be used in accordance with the invention.
• vectors of particular interest include prokaryotic and/or eukaryotic cloning vectors, expression vectors, fusion vectors, two- hybrid or reverse two-hybrid vectors, shuttle vectors for use in different hosts, mutagenesis vectors, transcription vectors, vectors for receiving large inserts (yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and PI artificial chromosomes (PACs)) and the like.
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• PACs PI artificial chromosomes
• vectors of interest include viral origin vectors (Ml 3 vectors, bacterial phage ⁇ vectors, baculovirus vectors, adenovirus vectors, and retrovirus vectors), high, low and adjustable copy number vectors, vectors which have compatible replicons for use in combination in a single host (e.g., pACYC184 and pBR322) and eukaryotic episomal replication vectors (e.g., pCDM8).
• the vectors contemplated by the invention include vectors containing inserted or additional nucleic acid fragments or sequences (e.g., recombinant vectors) as well as derivatives or variants of any of the vectors described herein.
• Expression vectors useful in accordance with the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids or bacteriophages, and vectors derived from combinations thereof, such as cosmids and phagemids, and will preferably include at least one selectable marker (such as a tetracycline or ampicillin resistance genes) and one or more promoters such as the phage lambda PL ⁇ ,. ⁇ ⁇ PCT/US00/04643 00/50625
• the preferred enzymatic method for making xNTPs from nucleotide monophosphates according to the present invention is represented schematically in Figure 1.
• the first step of the nucleotide synthesis involves converting the appropriate nucleotide monophosphate, xNMP, into the appropriate nucleotide diphosphate, xNDP, in the presence of one or more enzymes capable of converting the monophosphate into the diphosphate.
• the second step of the synthesis involves converting the nucleotide diphosphate, xNDP, into the desired nucleotide triphosphate, xNTP, in the presence of one or more enzymes capable of converting the diphosphate into a triphosphate.
• the first and second steps are accomplished in the presence of one or more phosphate donors which may be added to the reaction or prepared enzymatically during the reaction.
• the phosphate donors are different for the first and second steps of the reaction, although the same phosphate donor may be used.
• Phosphate donors used according to the present invention include any compound capable of donating one or more phosphates.
• the phosphate donor is PEP and xNTP.
• xNTP is the phosphate donor for the conversion of xNMP to xNDP and PEP is the phosphate donor for conversion of xNDP to xNTP.
• PEP is obtained enzymatically from the reaction of 3- PGA in the presence of the enzymes PGM and ENO and xNTP is obtained enzymatically from the reaction of xNDP in the presence of PYK and PEP.
• the nucleotide used when a nucleotide is used as a phosphate donor, the nucleotide used preferably is the desired nucleotide to be produced.
• the phosphate donor it is preferable to use xNTP and xNDP, respectively, as the phosphate donor. This reduces the likelihood that contaminating nucleotides will be present in the final reaction product.
• other high-energy phosphate donors can be used, including creatine phosphate, acetyl phosphate, arginine phosphate, and/or 1,3-dihosphoglycerate.
• 1,3-diphosphoglycerate can be synthesized enzymatically from gly cerol-3 -phosphate, phosphoric acid, and nicotinamide adenine dinucleotide (NAD), using the commercially available enzymes glycerol-3 -phosphate dehydrogenase, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase. If desired, NAD can be regenerated enzymatically using standard methods (for example, coupling the reaction with inexpensive pyruvate and lactate dehydrogenase).
• NAD nicotinamide adenine dinucleotide
• the reaction mix for xNTP synthesis using the 1,3-diphosphoglycerate method may include: xNMP, small amounts of xNTP and NAD, glycerol-3- phosphate, phosphoric acid, pyruvate, and one or more kinases, glycerol-3- phosphate dehydrogenase, triosephosphate isomerase, glyceraldehyde-3- phosphate dehydrogenase, and lactate dehydrogenase.
• the phosphate donor may be provided by starting at any point within the 1,3- diphosphoglycerate pathway by adjusting the starting substrates and enzymes accordingly (see Figure 2).
• succinyl CoA can be used as a phosphate donor co-substrate.
• Succinyl CoA can be made enzymatically from inexpensive citrate, using commercially available enzymes and NAD regeneration.
• the reaction mix using the succinyl CoA method may thus include: xNMP, small amounts of xNTP and NAD, Coenzyme A, citrate, pyruvate, and one or more kinases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl
• CoA synthetase, and lactate dehydrogenase are synthetase, and lactate dehydrogenase.
• PEP could be synthesized enzymatically from ribulose- 1,5-bisphosphate in bicarbonate buffer using one or more kinases, ribulose- 1,5-bisphosphate carboxylase, PGM, and ENO. Two equivalents of PEP may be generated from each equivalent of ribulose- 1,5-bisphosphate.
• phosphate donor may be provided by starting at any point within this pathway by adjusting the starting substrates and enzymes accordingly (see Figure 4).
• PEP can be synthesized from anywhere in the glycolytic pathway or any other pathway that yields PEP such as the Calvin cycle (i.e. ribulose), the aromatic amino acid biosynthesis of PEP, or any enzyme cascade leading to a generation of PEP (see Figure 5).
• Calvin cycle i.e. ribulose
• the aromatic amino acid biosynthesis of PEP or any enzyme cascade leading to a generation of PEP (see Figure 5).
• At least one of the phosphate donors should be higher energy than ATP in order to drive the reaction. If the phosphate donor has less energy than ATP, the reaction may reached equilibrium with the reactants, which may result in lower yield.
• any enzyme may be used. Such enzymes may be obtained commercially or may be isolated or purified from any source. If unavailable commercially, such enzymes may be isolated or purified from one or more host cells.
• recombinant host cells containing and expressing one or more genes encoding the desired enzyme is used as a source to isolate the desired enzymes. Recombinant techniques well known in the art may be used to clone and express the desired gene or genes into one or more vectors. The vectors may then be introduced into one or more host cells by standard transformation or transfection techniques well known in the art.
• the host cell producing the desired enzymes can be grown and harvested according to techniques well known in the art. Any culture medium containing nutrients that can be assimilated by the host cell can be used for growing the host cells. Optimal culture conditions can be selected according to the strain used in the composition of the culture medium.
• the desired enzymes can be purified by well known protein purification techniques. Such techniques may include extraction, precipition, chromatography, affinity chromatography, electrophoresis and the like. Assays to detect the presence of the desired enzymes during purification can be used to facilitate purification of these enzymes.
• the desired enzymes are prepared as fusion proteins which may facilitate purification.
• tags may include His-tags, GST tags, Trx-tags, epitope-tags (myc and HA), etc.
• Escherichia preferably E. coli
• E. coli Escherichia
• other sources of enzymes and the genes that code for them could be used.
• thermostable enzymes could be purified from a thermophilic bacterium, or the gene that codes for the enzyme could be cloned and expressed by standard recombinant techniques.
• sequences of the genes described herein could serve as hybridization probes to isolate a desired gene from a different host cell. The desired enzyme may then be purified by methods well known in the art as described above.
• Enzymes used in the invention may also be engineered, modified or mutated by standard techniques to improve their activity or to broaden their specificity for other substrates phosphate donors.
• an enzyme can be engineered to phosphorylate both xNMPs and/or xNDPs, using PEP as a phosphate donor or another phosphate donor ( Figure 6).
• an enzyme may also be designed to phosphorylate nucleosides as well, allowing the use of a less expensive starting material (Figure 7).
• an enzyme can be engineered to recognize only the xNMP ( Figure 8) and utilize PEP so that no catalytic xNTP would need to be added to the reaction.
• base specific enzymes and more preferably base specific kinases convert xNMPs into diphosphates.
• deoxyadenine monophosphate is converted to deoxyadenine diphosphate (dADP) by adenylate monophosphate kinase (AMK)
• deoxycytidine monophosphate is converted to deoxycytidine diphosphate (dCDP) by cytidine monophosphate kinase (CMK)
• deoxyguanosine monophosphate is converted to deoxyguanosine diphosphate (dGDP) by guanosine monophosphate kinase (GMK)
• deoxythymidine monophosphate is converted to deoxythymidine diphosphate (dTDP) by thymidine monophosphate kinase (TMK)
• guanosine monophosphate is converted to guanosine diphosphate (GDP) by guanosine monophosphate kinase (GMK), etc.
• conversion of the monophosphate is converted to deoxyadenine diphosphate (dADP) by adeny
• any enzyme capable of converting the diphosphate into the triphosphate may be used.
• a kinase is used and most preferably PYK is used to convert the diphosphates (e.g., xNDPs) into the desired triphosphate, (e.g., xNTPs).
• the phosphate donor for conversion of the diphosphate to the triphosphate is phosphoenolpyruvate (PEP). Since PEP is expensive, it is preferably synthesized enzymatically in a coupled reaction from D(-)3-phosphoglyceric acid (3-PGA) in the presence of the enzymes PGM and ENO.
• the complete reaction proceeds most efficiently as a coupled reaction with all enzymes present wherein the end product of the reaction is used as a catalyst in the first step of the reaction as shown in Figure 1.
• the reaction is "primed” with a small amount (catalytic amount) of product, in this case, xNTP, the reaction will accelerate as more catalyst, i.e. the end product, is made.
• the final reaction product is substantially pure and preferably is not contaminated with any undesired nucleotide such as ATP.
• the present invention relates to a method of making one or more xNMPs comprising (a) mixing one or more nucleosides or derivatives thereof, one or more enzymes capable of converting said nucleosides into xNMPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNMPs, wherein the nucleosides are preferably selected from the group consisting of adenine, cytidine, guanosine, thymidine and uridine or derivatives thereof. Any phosphate donor may be used.
• the enzyme used is preferably a kinase to allow phosphorylation of the nucleoside.
• the present invention also relates to a method of making one or more xNDPs comprising (a) mixing one or more xNMPs, one or more enzymes capable of converting said xNMPs into xNDPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNDPs.
• said xNMPs are preferably AMP, CMP, GMP, UMP, dAMP, dCMP, dGMP or dTMP, and derivatives thereof.
• the enzyme is preferably a kinase, most preferably a nucleotide monophosphate kinase (NMK), however, any kinase capable of phosphorylating a xNMP is suitable.
• the phosphate donor is preferably xNTP, but the invention also relates to the use of any phosphate donor that is capable of donating a phopsphate to an xNMP in order to convert it into a xNDP.
• the phosphate donor may be obtained separately and added to the reaction or may be produced enzymatically during the reaction.
• the present invention also relates to a method of making one or more xNTPs comprising (a) mixing one or more xNDPs, one or more enzymes capable of converting said xNDPs into xNTPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNTPs.
• the xNDP is preferably ADP, CDP, GDP, UDP, dADP, dCDP, dGDP or dTDP, and derivatives thereof.
• the enzyme is preferably a kinase, most preferably PYK, however any enzyme capable of phosphorylating xNDP is contemplated.
• the phosphate donor according to this embodiment is preferably PEP, but any phosphate donor capable of donating a phosphate to xNDP to permit the conversion to xNTP is suitable.
• the phosphate donor may be obtained separately and added to the reaction or may be made during the reaction by enzymatic synthesis. Enzymatic synthesis includes synthesis by mixing 3-PGA with PGM and ENO or by mixing 2-PGA with ENO.
• any synthesis reaction which produces a phosphate donor may be used according to the present invention.
• the present invention also includes a method of making one or more xNDPs comprising (a) mixing one or more nucleosides or derivatives thereof, one or more enzymes capable of converting said nucleosides into xNMPs and converting said xNMPs into xNDPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNDPs.
• Another preferred embodiment includes a method of making one or more xNTPs comprising (a) mixing one or more xNMPs, one or more enzymes capable of converting said xNMPs into xNDPs and converting said xNDPs into xNTPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make the desired xNTPs.
• Yet another preferred embodiment concerns a method of making one or more xNTPs comprising: (a) mixing one or more nucleosides or derivatives thereof, one or more enzymes capable of converting said nucleosides into xNMP, converting said xNMPs into xNDPs, and converting said xNDPs into xNTPs, and one or more phosphate donors; and (b) incubating said mixture under conditions sufficient to make said xNTPs.
• nucleotides produced by the methods of the invention may be purified and isolated by techniques well known in the art. Purification techniques include chromotography, preferably by high pressure liquid chromatography (HPLC) and/or ion exchange chromotography. Purification or isolation of such nucleotides may provide for substantially pure nucleotides in accordance with the invention.
• Purification techniques include chromotography, preferably by high pressure liquid chromatography (HPLC) and/or ion exchange chromotography. Purification or isolation of such nucleotides may provide for substantially pure nucleotides in accordance with the invention.
• the present invention permits the synthesis of high quality nucleotides which may be used in amplification (PCR), sequencing, labeling, nucleic acid synthesis, and cDNA synthesis.
• the nucleotides of the invention may be included in kits to perform such molecular biology techniques.
• Preferred conditions for carrying out the methods of the invention include incubation at a temperature of about 10 to about 60°C, preferably from about 20 to about 45 °C and most preferably at about 37°C. Further preferred reaction conditions include incubating the reaction at a pH of about 5.0 to about 9.0, preferably from about 6.0 to about 8.5, preferably from about 7.0 to about 8.0, and most preferably from about 7.2 to about 7.8. It will be understood that one skilled in the art can optimize the conditions to accomplish the desired enzymatic reaction and obtain the desired product. Such conditions may vary depending on the substrates, the enzymes and the desired reaction.
• the present invention also relates to a composition
• a composition comprising (a) one or more nucleosides or derivatives thereof; (b) one or more enzymes capable of converting said nucleosides into xNMPs; and (c) one or more phosphate donors.
• the nucleosides are adenine, cytidine, guanosine, thymidine or uridine or derivatives thereof.
• Such compositions may further comprise in place of or in addition to the phosphate donors, one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the present invention further relates to a composition
• a composition comprising (a) one or more xNMPs; (b) one or more enzymes capable of converting said xNMPs into xNDPs; and (c) one or more phosphate donors.
• the xNMPs are AMP, CMP, GMP, UMP, dAMP, dCMP, dGMP or dTMP or derivatives thereof.
• the enzyme is preferably a kinase, more preferably NMK
• the phosphate donor is preferably xNTP.
• Such compositions may further comprise in place of or in addition to the phosphate donors, one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the present invention further relates to a composition
• a composition comprising (a) one or more xNDPs; (b) one or more enzymes capable of converting said xNDPs into xNTPs; and (c) one or more phosphate donors
• said xNDPs are preferably ADP, CDP, GDP, UDP, dADP, dCDP, dGDP or dTDP or derivatives thereof.
• the enzyme is a kinase, more preferably PYK
• the phosphate donor is preferably PEP.
• Such compositions may further comprise in place of or in addition to the phosphate donors, one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the composition preferably comprises 3-PGA in the presence of PGM and ENO or 2-PGA in the presence of ENO in place of or in addition to PEP.
• the present invention further relates to a composition comprising (a) one or more nucleosides or derivatives thereof; (b) one or more enzymes capable of converting said nucleosides into xNMPs and converting said xNMPs into xNDPs; and (c) one or more phosphate donors.
• the present invention relates to a composition
• a composition comprising (a) one or more xNMPs; (b) one or more enzymes capable of converting said xNMPs into xNDPs and converting said xNDPs into xNTPs; and (c) one or more phosphate donors.
• the invention further relates to a composition comprising (a) one or more nucleosides or derivatives thereof; (b) one or more enzymes capable of converting said nucleosides into xNMPs, converting said xNMPs into xNDPs, and converting said xNDPs into xNTPs; and (c) one or more phosphate donors.
• Such compositions may further comprise in place of or in addition to the phosphate donors, one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• compositions comprising one or more enzyme capable carrying out the methods of the invention.
• Such composition may comprise at least one enzyme capable of converting one or more nucleosides or derivatives thereof into xNMPs and at least one enzyme capable of converting xNMPs into xNDPs.
• Such composition may also comprise at least one enzyme capable of converting xNMPs into xNDPs and at least one enzyme capable of converting xNDPs into xNTPs.
• Preferred enzymes in such compositions include any kinase and preferably include
• composition may further comprise one or more phosphate donors and/or one or more enzymes capable of converting a phosphate donor substrate into a phosphate donor.
• enzymes for synthesizing phosphate donors include PGM and/or ENO.
• the present invention further relates to a composition comprising one or more nucleosides or derivatives thereof and/or one or more nucleotides, and at least one phosphate donor, wherein the nucleotides are preferably xNMP or xNDP and the phosphate donor is preferably xNTP or PEP.
• compositions may further comprise in place of or in addition to the phosphate donors, one or more phosphate donor substrates and one or more enzymes capable of converting such substrates into phosphate donors.
• the present invention also relates to kits.
• kits comprising one or more components for making nucleotides in accordance with the invention.
• Such kits may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as vials, test tubes and the like.
• container means comprises components or a mixture of components needed to perform nucleotide synthesis.
• kits for the enzymatic preparation of nucleotides may comprise at least one component selected from the group consisting of at least one enzyme for nucleotide synthesis, preferably a kinase such as NMK and/or PYK, at least one enzyme for phosphate donor synthesis, at least one phosphate donor such as PEP, at least one phosphate donor substrate, at least one substrate for nucleotide synthesis (e.g., nucleosides, xNMP or xNDP), reaction buffers, instructions, etc.
• at least one enzyme for nucleotide synthesis preferably a kinase such as NMK and/or PYK
• at least one enzyme for phosphate donor synthesis at least one phosphate donor such as PEP
• at least one phosphate donor substrate e.g., nucleosides, xNMP or xNDP
• reaction buffers e.g., nucleosides, xNMP or xNDP
• a 5-ml aqueous reaction was prepared containing 33 mM Tris-Cl pH 7.5, 30 mM KC1, 30 mM MgCl 2 , 4 mM DTT, 60 mM 3-phosphoglyceric acid (3-PGA), 30 mM dAMP and 1 mM ATP or dATP.
• the following enzymes were added: 89 units phosphogly cerate mutase (PGM), 52 units enolase (ENO), 14 units adenosine monophosphate kinase (AMK) and 54 units pyruvate kinase (PYK).
• the enzymes were assayed using a modified method further described below.
• the reaction was incubated at room temperature for 3 days.
• the synthesis of dATP was confirmed using thin layer chromatography (TLC) on poly(ethyleneimine) plates in 10 mM Tris at a pH 7.5 and 1M LiCl.
• Reaction products were analyzed by capillary electrophoresis (CE) (uSIL-WAX capillary, J&W Scientific, 37 cm x 50 ⁇ ; 50 mM triethylammonium acetate pH 5.2; 11 kV; reverse polarity; 2 sec pressure injection; analysis at 254 nm) and by HPLC (using standard ion-pair chromatographic methods). Purity of crude dATP synthesized by this method was found to be >88%.
• CE capillary electrophoresis
• FIG. 9 shows the pH profile of the first 30 minutes of reaction.
• the pH rise is due to H + consumption during the PYK reaction.
• the AMK reaction is limiting, as very little dATP is present.
• the overall reaction accelerates as the AMK reaction becomes more efficient.
• the pH controller begins to deliver HCl to the reaction and the pH is maintained at 7.8.
• MgCl 2 1.0 mM DTT, 20 mM 3-PGA, 10 mM dGMP and 0.025 mM ATP.
• the following enzymes were added: 30,000 units PYK, 4000 units PGM, 900 units ENO and 6000 units guanosine monophosphate kinase (GMK).
• the reaction was incubated at 37°C with pH controlled below 7.8 with 1M acetic acid. An additional 30 mmol 3-PGA were added after 2.5 h of incubation.
• Results were analyzed by HPLC as described herein and crude dGTP synthesized by this method was obtained in 92% yield after 5 h of reaction. dGTP was found to separate well from ATP by standard anion exchange chromatography processing methods.
• GMK was replaced by AMK using the same reaction conditions, but the yield was significantly lower.
• dGTP was replaced with ATP as a catalyst using the same reaction conditions. Although the yield was not as good as the results obtained using dGTP as a catalyst, the results were significant. See Figure 10.
• Figure 12 shows the effect of the 3-PGA/dTMP ratio on the dTTP synthesis reaction. In order to achieve a >90% yield, >3 equivalents of 3-PGA are required.
• Table 1 summarizes the results of dNTP synthesis reactions using the methods of the present invention as described above.
• Table 2 summarizes the enzymatic requirements for dNTP synthesis according to the present invention.
• the following enzymes are added: 1500 units/ml PYK, 200 units/ml PGM, 45 units/ml ENO and 300 units/ml adenosine monophosphate kinase (AMK).
• the reaction is incubated at 37°C with pH controlled below 7.8 with IM acetic acid.
• the total reaction time is 5 h.
• FIG. 13 represents an analysis of the reaction product of the enzymatic synthesis of dATP product using ATP as a catalyst.
• Table 3 a presents the peak number, retention time (in minutes), and area (expressed as a percentage) for each peak present in the HPLC chromatogram depicted in Figure 13: Table 3a
• Figure 14 represents an analysis of the purified product of the enzymatic synthesis of dATP product using ATP as a catalyst.
• Table 3b presents the peak number, retention time (in minutes), and area (expressed as a percentage) for each peak present in the HPLC chromatogram depicted in Figure 14:
• the purity of the dATP by HPLC was 92.9%.
• the HPLC analysis of the purified dATP showed a contaminant of >2% (3.1%) that eluted as ATP.
• the column was a TosoHaas Super Q-650(s) with a 5.0 ml packed bed of resin, equilibrated to Buffer A, 200 mM Triethylamine Formate (pH 5.10) in H 2 O.
• the column was washed using 50 ml Buffer A at a flow of 100 cm/hr (3.3 ml/min).
• the gradient was a linear 100 ml gradient from Buffer A to Buffer B, 1.0 M Triethylamine Formate (pH 5.12), in 1.5 ml fractions at a flow of 30 cm/hr (1 ml/min). All gradient peak fractions showing > l A peak height were pooled.
• Table 3c summarizes the major peaks seen in Figures 13 and 14. As seen in Figure 13, the purified dATP synthesized using ATP as a catalyst showed a contamination that eluted as ATP.
• Figure 15 represents an analysis of the reaction product.
• Table 4a presents the peak number, retention time (in minutes), and area (expressed as a percentage) for each peak present in the HPLC chromatogram depicted in Figure 15: 625
• Figure 16 represents an analysis of the purified reaction product.
• Table 4b presents the peak number, retention time (in minutes), and area (expressed as a percentage) for each peak present in the HPLC chromatogram depicted in Figure 16: Table 4b
• the purity of the dATP by HPLC was 94.2%.
• the HPLC analysis of the purified dATP did not show any prominent contamination that eluted as ATP.
• the column was a TosoHaas Super Q-650(s), with a 5.0 ml packed bed of resin and equilibrated to Buffer A.
• the column was washed using 50 ml Buffer A at a flow of 100 cm/hr (3.3 ml/min).
• the gradient was a linear 100 ml gradient from Buffer A to Buffer B in 1.5 ml fractions at a flow of 30 cm/hr (1 ml/min). All gradient peak fractions showing > X A peak height were pooled.
• Table 4c summarizes the peaks seen in Figures 15 and 16. As seen in Figure 16, the purified dATP synthesized using dATP as a catalyst showed no contamination that eluted as ATP.
• dNTP as a catalyst for the synthesis of dNTP according to the present invention clearly eliminates the problems associated with ATP contamination.
• Nucleotide monophosphate kinases were assayed as follows: For adenosine monophosphate kinase, the reaction mix (in a 1-ml disposable spectrophotometer cuvette) included 50 M Tris-Cl pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 2 mM PEP, 0.2 mM NADH, 0.2 mM dAMP, 0.2 mM ATP, 5 units/ml lactate dehydrogenase and 8 units/ml PYK. Reactions were equilibrated at 37°C and initiated by the addition of enzyme.
• Enzyme activity was quantified by following the oxidation of NADH at 340 nm in a spectrophotometer.
• One unit of enzyme is defined as the amount of AMK required to oxidize 1 mmol of NADH in 1 min at 37°C.
• Cytidine monophosphate kinase and thymidine monophosphate kinase were assayed similarly, except that dCMP and dTMP, respectively, replaced dAMP in the reaction mix.
• the production of high levels of PYK for dNTP synthesis benefits the NMK assay because it allows high levels of this coupling enzyme to be added to the reaction.
• Blondin, et al, (1994) Analytical Biochemistry 220; 219-221 have shown that the substrate specificity of PYK for nucleotides such as dCMP requires high levels of PYK (>5 units/ml) to be added to the NMK assay.
• the addition of another enzyme such as nucleoside diphosphate kinase is recommended.
• the method of the present invention permits the purification of over 1,000,000 units of PYK from cells (final enzyme concentration: 1400 U/ml).
• the addition of excess CMK leads to a rapid oxidation of NADH, showing that PYK and
• Pyruvate kinase was assayed similarly to AMK except that the reaction mix contained 0.2 mM ADP instead of dAMP and ATP and only LDH was used as a coupling enzyme. PYK enzyme was added to the reaction mix prior to pre-equilibration and the reaction was initiated by the addition of ADP.
• Enolase was assayed as follows: the reaction mix (in a 1-ml quartz spectrophotometer cuvette) included 50 mM Tris-Cl pH 8.0, 50 mM KCl, 10 mM MgCl 2 , 1 M DTT, 2 mM D-(+)-2-phosphoglyerate. Reactions were equilibrated at 37°C and initiated by the addition of enzyme. Enzyme activity was quantified by following the formation of PEP at 240 nm in a spectrophotometer. The extinction coefficient of PEP is 1510 M 'c "1 at pH 8. One unit of enzyme is defined as the amount of enolase required to form 1 mmol of PEP in 1 min at 37°C.
• Phospho gly cerate mutase was assayed similarly to enolase except that D-(+)-2-phosphoglycerate was omitted from the reaction mix and was replaced with 2 mM 3-phosphoglycerate, 0.2 mM 2,3-diphosphoglycerate, and 2 units/ml enolase.
• the genes for the 4 E. coli enzymes that convert dNMP to dNDP (AMK, CMK, GMK and TMK) and the genes for the E. coli enzymes PYK, PGM and ENO were cloned from E. coli DNA by
• AMK E. coli adenine monophosphate kinase
• the DNA sequence of the gene for E. coli adenine monophosphate kinase (AMK) has been published by Brune, et al. (1985) Nucleic Acids Research 13 (19), 7139-7151. According to Brune, et al, the gene is referred to as the adk (adenylate kinase) gene. However, for simplicity, a uniform nomenclature which is consistent for all 4 monophosphate kinase genes and proteins has been chosen. Two oligonucleotides were synthesized to clone the AMK gene by a standard PCR regimen:
• Each oligonucleotide has a 12 base 5' extension, containing 4 uracil residues each, which allow for simple cloning of the PCR fragment by the
• the insert was removed with Ndel-Sstl and subcloned into the Ndel- Sstl sites of pROEXl, forming pROEX-AMK (see Figure 19).
• the His tag is marked on the map but is not labeled because it is so small.
• TMK coli thymidine monophosphate kinase
• CMK cytidine monophosphate kinase
• GMK guanosine monophosphate kinase
• PGM phoshoglycerate mutase
• ENO pyruvate kinase
• the TMK, CMK, GMK, PGM, PYK and ENO genes were cloned by PCR (see Figures 20, 22, 24, 26, 28 and 30 respectively).
• CMK, GMK, PGM and ENO were subcloned into an expression vector which added a histidine tag in the same way that the AMK gene was cloned and subcloned (see Figures 23, 25, 27 and 31 respectively).
• TMK was cloned into pROEXl-HT with Ehel and Sstl instead of Ndel-Sstl (.see Figure 21).
• PYK the histidine tag was added to the carboxy end of the protein (see Figure 29).
• the E. coli cells with these plasmids were then induced for expression of the histidine-tagged protein, and the proteins were purified on columns which bind histidine-tagged proteins, in the same way that AMK was expressed and purified as described above.
• GENBANK LOCUS ECU41456, ACCESSION: U41456 See, Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding TMK locus, J.
• Oligonucleotides to clone TMK by PCR (Ehel site in bold):
• CMK gene encoding cytidine monophosphate kinase is located in the rpsA operon and is required for normal replication rate in Escherichia coli, J. Bacteriol. 177 (3), 517-523 (1995) and Oshima et al., A 718-kb DNA sequence of the Escherichia coli K- 12 genome corresponding to the 12.7-28.0 min region on the linkage map, DNA Res. 3 (3), 137-155 (1996).
• each oligonucleotide has a 12 base 5' extension, containing 4 uracil residues each, which allow for simple cloning of the PCR fragment by the UDG cloning method.
• an Ndel endonuclease site (bold) is constructed out of the ATG start codon of each gene (except TMK, which uses an Ehel site).
• an Sstl site (bold) is added just after the termination codon (italic).
• PYK a histidine tag at the amino end was found to be unsuitable, so a histidine coding sequence was - ⁇ 2-
• the insert was subcloned in pROEXl, placing the appropriate gene downstream from the Trc promoter in pROEXl (except for PYK), and adding a histidine coding sequence to the amino end of the coding sequence so that the recombinant protein could be purified by using a nickel column.
• PYK since the histidine sequence was added to the carboxy end of the gene, and the histidine-tagged gene was cloned into the Ndel-Sstl sites of expression vector pTrcN, forming pTrcN-PYK-CHis (Figure 29).
• E. coli strain DHlOB/pRO ⁇ X-AMK harbors the pRO ⁇ X-AMK plasmid, and thus expresses recombinant, histidine-tagged AMK when the Trc promoter is activated with IPTG.
• DHlOB/pRO ⁇ X-AMK cells were grown in 200 ml CG + 0.01% PPG 100 ⁇ g ampicillin/ml at 37°C until they reached an OD 590 nm of 1. IPTG was added to a final concentration of 1 mM. After 3 more hours, cells were harvested by centrifugation, and the cell pellet was stored at -20°C until used.
• the pellet was thawed with 20 ml of cold (4°C) cracking buffer (described below), and 20 mg of lysozyme was added. The following steps were preformed at 4°C.
• the cells were broken by ultra- sonication, and the lysate was centrifuged for 60 min at 10,000 rpm in a sorvall SS34 rotor. The supernatant was applied to a 15 ml Nickel-NTA column and the column was washed with 150 ml of Buffer ⁇ (described below). His-tagged AMK was eluted with 150 ml of a linear gradient of imidazole from 20 mM to 400 M, accomplished by appropriate mixing of Buffer ⁇ and Buffer F (described below).
• DHlOB/pROEX-CMK, DHlOB/pROEX-GMK DHlOB/pROEX- PGM, and DHlOB/pROEX-ENO cause the expression of histidine-tagged
• CMK, GMK, PGM, and ENO proteins were similarly expressed from DHlOB/pTrcN-PYK-CHis.
• DH10B cells carrying the appropriate plasmid were grown, induced, further grown, harvested, broken by ultra-sonication, and centrifuged as described above for histidine-tagged AMK. The supernatants were applied to 15 ml Nickel-NTA columns and chromatographed as described above for histidine-tagged AMK.
• His-tagged proteins eluted at imidazole concentrations from 125 mM to 175 mM imidazole, (except for GMK, which eluted from about 165 mM to 215 mM imidazole) and were detected by protein concentration determination and by polyacrylamide gel electrophoresis. Fractions containing the appropriate His- tagged enzyme were pooled and dialyzed against Buffer G at 4°C. His-tagged proteins were stored at -20°C in Buffer G.
• the PCR regimen included 100 ⁇ l Supermix, 1 ⁇ l 100 mM each oligonucleotide, 1 ⁇ l 200 ng/ ⁇ l target DNA and 1 ⁇ l (5 U) additional recombinant Taq DNA polymerase:
• the preparation of the Nickel-NTA column included charging a 15 ml bed of NTA-sepharose (Pharmacia) with 75 ml of 200 mM NiSO4, washing with water, washing with Buffer F, and then washing with Buffer E.
• CA capsule per 25 ml water, autoclaved; IPTG: 200 mM in water, filter sterilized, stored at -20°C until used.

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