Taq purification


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Taq purification

  1. 1. ANALYTICAL BIOCHFMISTRY 19 1,396-400 (1990)Purification of Thermus aquaticus DNA PolymeraseExpressed in Escherichia co/iDavid R. Engelke, *J Alexandra Krikos,? Mary E. Bruck,t$ and David Ginsburgt*$$Departments of *Biological Chemistry, THuman Genetics,$Internal Medicine, and *The Howard HughesMedical Institute, the University of Michigan, Ann Arbor, Michigan, 48109Received July 13, 1990DNA polymerase from Thermus aquaticus has be-come a common reagent in molecular biology because ofits utility in DNA amplification and DNA sequencingprotocols. A simplified method is described here for iso-lating the recombinant Taq enzyme after overproduc-tion in Escherichia coli. Purification requires 8 to 10 hand entails heat treating and clearing the E. coli lysate,followed by precipitation of the enzyme with polyethy-leneimine and elution from Bio Rex 70 ion exchangeresin in a single salt step. The resulting enzyme prepara-tion contains a single, nearly homogeneous protein con-sistent with the previously established size of the TaqDNA polymerase in a yield of 40-60 mg of protein perliter of cell culture. 0 1990 Academic Press, IIIC.In recent years experimental possibilities have beenradically affected by the advent of selective DNA seg-ment amplification in vitro using the polymerase chainreaction [PCR, (l)]‘. Central to the wide disseminationof these techniques has been the introduction of heatstable DNA polymerases, primarily from Thermusaquaticus (Tuq), so that the process can be automatedusing a thermal cycler without continuous addition offresh DNA polymerase (2). Methods for cloning, directsequencing, clinical diagnosis, and myriad other uses (3)have proliferated with the ability to acquire microgramquantities of particular DNA fragments from mixturesas complex as whole genomes and present in quantitiesas low as the DNA or RNA from a single cell.Since these combined applications often result inlarge expenditures of enzyme, it is of some interest to1 To whom correspondence should be addressed.’ Abbreviations used: PCR, polymerase chain reaction; IPTG, iso-propyl-1-thio-,9-D-galactopyranoside; PMSF, phenylmethylsulfonylfluoride; PEI, polyethyleneimine.396have a method by which individual laboratories can eas-ily purify large quantities of the Tuq DNA polymerasefor routine use. We describe here the use of an overpro-ducing plasmid construct of the Tuq DNA polymerasegene with a simple l-day purification scheme to yieldapproximately 1 g of nearly homogenous enzyme from al%-liter culture of Escherichiu coli.MATERIALS AND METHODSStrains and culture media. T. aquaticus strain YT-1was the gift of D. Hunt and N. Pace (Indiana Univer-sity) and was grown at 5560°C on solid medium con-taining 1.5% agar and nutrients described previously(4). E. coli strain DHl, used as a host for recombinantplasmids to overproduce the Tuq DNA polymerase, wasgrown in LB medium supplemented with 80 pg/ml am-picillin to propagate plasmids and indicated concentra-tions of isopropyl-1-thio-/3-D-galactopyranoside (IPTG)to induce production of the enzyme.Activity and protein assays. Protein concentrationswere determined by measuring absorbance ratios at 280and 260 nm. Protein content was visualized by electro-phoresis through denaturing polyacrylamide gels [5%stacking gel, 10% separating gel, (5)] and staining withCoomassie brilliant blue R. DNA polymerase assaysduring purification were performed by monitoring incor-poration of radiolabeled dATP into high molecularweight DNA using a primed Ml3 viral template. Briefly,1~1 of serially diluted enzyme fraction was assayed in afinal volume of 5 ~1 containing 50 InM Hepes (pH 7.9)(N-2 hydroxyethylpiperizine-N’-2-ethanesulfonic acid),1.5 mM MgCl,, 50 mM KCl, 100 I.IM each dGTP, dCTP,and dTTP, 10 PM dATP including 1 &i [(w-~‘P]~ATP, 1pg M13mp18 single-stranded DNA, and 0.1 pg Ml3 uni-versal primer 17mer. Reactions were incubated for 1min at 37°C and then for 3 min at 65°C. Deionized form-amide (10 ~1) containing 0.1% xylene cyan01 was addedto terminate the reactions and the samples were heated0003-2697190 $3.00Copyright 0 1990 by Academic Press, Inc.All rights of reproduction in any form reserved
  2. 2. PURIFICATION OF Thermus aquaticus DNA POLYMERASE 397TABLE 1Taq DNA Polymerase Purification SummaryProtein Nucleic acidEnzyme fraction (md bd Activity”Cleared lysate 8460 1020 7 x lo6PEI eluate 2640 48 7 x lo6BioRex 70 eluate 1140 nd” 6 X 10’Note. Protein, nucleic acid and enzyme activity content is given formajor fractions of the isolation. Assay procedures are described underMaterials and Methods.a Enzyme activity approximated by comparison to commercial en-zyme in both primed Ml3 and PCR assays.* None detectable.before analysis of 5 ~1 aliquots by electrophoresisthrough denaturing 8% polyacrylamide gels (6) and au-toradiography for 5-60 min using DuPont Cronex 4film. Titration of enzyme fractions using polymerasechain reaction amplification from human genomic DNAwas performed in 50-~1 reactions with 1 ~1 of dilutedenzyme added immediately before overlaying with min-eral oil and beginning temperature cycling. Reactionscontained 50 mM Hepes (pH 7.9), 1.5 mM MgCl,, 50 mMKCI, 100 PM dATP, dGTP, dCTP, and dTTP, 0.6 pghuman total DNA, and 0.1 pg each of two 35mer primers(5’-CTTCAGCGAGGCACAGTCCAAAGGGGACATC-CTGC-3’ and 5’-CATGACGTCCACAAGGCTCAGC-AAATGGGCTTTCT-3’) that amplify a 2100-base pairfragment of the human von Willebrand factor genomicDNA including portions of exons 28-30 (8,9). Reactionswere cycled 35 times for 1.25 min at 94°C and 3 min at72°C. Aliquots of 5 ~1were analyzed by electrophoresisthrough 1.2% agarose gels in the presence of TBE buffer[90 mM Tris-HCl, pH 8.3,90 mM borate, 25 mM EDTA(ethylenediaminetetracetate)] and 0.3 pg/ml ethidiumbromide using X phage DNA cleaved with B&E11 (Be-thesda Research Laboratories) as size markers. Sincethe most common practical uses for this enzyme areprimer extension sequencing and PCR, enzyme activitywas determined by multiple titrations with both theprimed Ml3 assay and the PCR assay relative to com-mercial Tuq DNA polymerase (Cetus Corp.). Enzymeactivity given in Table 1 is expressed as previously de-fined units (7).Construction of overproducing clone. T. aquaticusDNA was isolated from duplicate individual colonies ofstrain YT-1 as described (7) except that the colonieswere suspended in only 200 ~1 of lysis buffer and thepreparation was scaled down proportionately. A2500-base pair fragment containing the Taq DNA poly-merase gene was prepared by PCR amplification from0.1,0.2, or 0.5 pg of this DNA template (either uncut orafter cleavage with BstEII) and 0.2 pg each of twoprimers that annealled to opposite ends of the codingregion. The 5’ (amino terminal) primer sequence was5’-CACGAATTCGGGGATGCTGCCCCTCTTTGAG-CCCAAG and the 3’ (carboxyl terminal) primer was5’-GTGAGATCTATCACTCCTTGGCGGAGAGCCA-GTC, with the first 9 nucleotides in each case being ex-trinsic to the Tuq DNA polymerase gene and creatingthe underlined unique EcoRI and BglII restriction endo-nuclease sites, respectively, at the two ends of the am-plified DNA fragment. Thirty-five-cycle PCR reactionswere performed under standard buffer conditions (3)with alternating incubations of 94°C for 1min and 72°Cfor 3 min, ending with one lo-min incubation at 72°C.The resulting amplification products were cleaved withEcoRI and BgZII and DNA fragments of approximately2500 bp were isolated from low melting point agarosegels. The fragments were ligated into expression vectorpTTQl8 [Amersham, (lo)] that had been cleaved withEcoRI and BamHI, giving a fusion that usesthe tat pro-moter, ribosome binding site, and transcription termin-ator from the vector. The native Tuq translation ter-minator is present, but the fusion uses the initiatormethionine of the vector, replacing the first two aminoacids encoded for the native enzyme (Met-Arg- * * *)with Met-Asn-Ser-. . . (7). Recombinant plasmidswere transformed into E. coli strain DHl and plated onLB media with ampicillin. Correct plasmid construc-tions were initially identified by restriction endonucle-aseanalysis of plasmid minipreps (11) and confirmed byinducing logarithmic l-ml cultures for 4 h with 2 mMIPTG and testing lysates (small scale version of clari-fied lysates described below) for Taq DNA polymerase.Induced overproduction of the expected 94-kDa protein(7) was monitored with denaturing polyacrylamide gelelectrophoresis (5) and activity in diluted lysates wasmeasured with the primed Ml3 template assay.Purification of Taq DNA polymerase. Large scalepreparations of the Taq DNA polymerase were donefrom 12-liter batches of cells. Preparation of cell lysateswas performed by minor modifications of a previouslydescribed method (7). LB media plus ampicillin was in-noculated with 1 ml of a saturated overnight culture ofDHl containing the recombinant plasmid and grown at37°C with good aeration to early exponential phase(A 600nmapproximately 0.2). IPTG was added to a finalconcentration of 0.5 mM and the cultures were allowedto continue growing for 16-20 h. IPTG titrations andtime courses of induction showed no difference in thelevel of accumulated Taq protein with concentrations aslow as 0.25 mM IPTG and induction times of 6 to 24 h(data not shown). Cells were harvested, resuspended in2400 ml buffer A (50 mM Tris-HCl, pH 7.9,50 mM dex-trose, 1 mM EDTA), collected by centrifugation, resus-pended in 480 ml buffer A containing 4 mg/ml lysozyme(Sigma), and incubated at room temperature for 15min.Buffer B (480 ml) was then added [10mM Tris-HCl, pH
  3. 3. 398 ENGELKE ET AL.7.9, 50 mM KCl, 1 mM EDTA, 1 mM phenylmethylsul-fonyl fluoride (PMSF), 0.5% Tween 20, 0.5% Nonidet-P40) and the mixture was incubated in a 75°C waterbath for 60 min in 180-ml aliquots to allow more rapidtemperature equilibration. Cell debris and denaturedprotein was removed by centrifugation at 4°C for 15 minin a Sorvall GSA rotor at 8000 rpm. Subsequent opera-tions were performed at 4 to 8°C.Taq DNA polymerase was recovered from this clari-fied lysate by precipitation with polyethyleneimine(PEI). Because the required PEI concentration wasslightly variable and too much PEI inhibited precipita-tion, preliminary titrations were normally performed.Neutralized 10% PEI (Miles) was added to 200-~1 ali-quots of lysate to a final concentration of 0.05 to 0.8%and the mixture was vortexed and incubated for 5 min atroom temperature. The precipitate was recovered byspinning for 5 min in a microcentrifuge and resus-pended in 200 ~1 buffer C (20 mM HEPES, pH 7.9,l mMEDTA, 0.5 InM PMSF, 0.5% Tween 20,0.5% Nonident-P40) containing 0.25 M KC1 to elute the DNA polymer-ase. Residual precipitate was removed by recentrifuga-tion and activity in dilutions of the supernatant wasdetermined by the primed Ml3 assay. After titration,neutralized 10% PEI was added dropwise to the remain-ing lysate to the optimum concentration (usually0.15%), incubated 10 min, and the precipitate collectedby centrifugation for 20 min at 8000 rpm in a SorvallGSA rotor. The pellet was washed once by resuspensionin 240 ml buffer C containing 0.025 M KC1 and the pre-cipitate was recovered by centrifugation. The Taq DNApolymerase was eluted from the precipitate by resuspen-sion in 240 ml buffer C containing 0.15 M KC1 and theresidual precipitate was removed by centrifugation. Re-suspensions of the PEI precipitates were routinely donewith several strokes in a Dounce homogenizer with aloose pestle to ensure an even suspension.The PEI eluate was diluted to 50 mM KC1 with bufferC and applied to a 150-ml column of BioRex 70 ion ex-changer (Bio-Rad) that had been preequilibrated inbuffer C containing 50 mM KCl. The column was thenwashed with 6 column volumes of the same buffer andthe enzyme was eluted in a single step with buffer Ccontaining 200 mM KCl. The peak of protein from thisstep was pooled and dialyzed for 12 h against twochanges of 1 liter each storage buffer (20 mM HEPES,pH 7.9, 100 mM KCl, 0.1 InM EDTA, 0.5 InM PMSF, 1mM dithiothreitol, 50% glycerol). No gelatin is added tothis buffer (7) because the DNA polymerase is suffi-ciently concentrated for storage (lo-20 mg/ml). Thispreparation is stable for at least 10 min at 94”C, at least24 h at room temperature, and at least 6 rounds of freez-ing and thawing (data not shown). Aliquots of 100 ~1arekept at -80°C except for working stocks, which arestable for at least several months when stored at -10°C.Taqpol -FIG. 1. Analysis of protein content. Denaturing polyacrylamide gelelectrophoresis with Coomassie staining was performed as describedunder Materials and Methods to visualize the protein in aliquots fromseveral stages of the purification. The two right-hand lanes containedclarified lysate from DHl cells containing the overproducer plasmideither with or without IPTG induction of Taq DNA polymerase syn-thesis. The left-hand two lanes show the PEI eluate and final enzymefraction (BioRex 70 eluate), and have been deliberately overloadedwith 40 pg of protein per lane. The expected position of full-lengthTuq DNA polymerase (94 kDa) relative to standard markers (notshown) is indicated to the left.RESULTS AND DISCUSSIONPurification of Taq DNA polymerase is relativelystraightforward when it is produced in E. coli becausethe enzyme appears to accumulate stably on long-terminduction and because the initial steps of heat treatingand clarifying the cell lysate denature and remove mostcontaminating protein. A summary of the recovery ofprotein and DNA polymerase activity is presented inTable 1 and denaturing polyacrylamide gel visualizationof protein fractions from the isolation procedure isshown in Fig. 1. Comparison of the induced and unin-duced pattern of proteins in the clarified lysates shows
  4. 4. PURIFICATION OF Thermus aquuticlls DNA POLYMERASE 399Commercial BioRex 70 EluateCommercial BioRex 70 EluateFIG. 2. Tao DNA polymerase assays. Activity of the isolated Tag DNA polymerase was titrated relative to a commercial preparation usingboth single-round primer extension assays and polymerase chain reaction amplifications. (A) One microgram of Ml3mplS viral DNA wasprimed with the universal forward primer and the incorporation of [a-32P]dATP into high molecular weight primer extension products withdilutions of the indicated enzymes was monitored by denaturing polyacrylamide gel electrophoresis and autoradiography. (B) Thirty-fivecycles of PCR were performed with total human DNA and the primers described under Materials and Methods using the same dilutions ofenzymes as in A. The position of the expected amplified DNA fragment relative to BstEII-cleaved X phage DNA markers is indicated by anarrow. Disappearance of correctly amplified fragment at high enzyme concentrations is discussed in the text.that a significant proportion of the protein in lysatesfrom induced cells appears as a band corresponding tothe full-length Tuq DNA polymerase (94 kDa) and thatPEI precipitation of the enzyme removes most contami-nants. The final BioRex 70 chromatography step re-moves slightly more extraneous protein, but more im-portantly removes residual PEI that can interfere withDNA polymerase function. The final enzyme fractionstill contains trace contamination from E. coli proteins(Fig. 1). Residual E. coli nucleic acids are undetectableby absorbance at 260 nm but can be detected by moresensitive assays at levels consistent with contamina-tions in commercial preparations (Norman Pace, per-sonal communication).Although the Taq DNA polymerase produced bythese methods has performed well in a wide variety ofapplications, it should be emphasized that it is abso-lutely necessary to carefully titrate the correct dilutionof the enzyme, especially for use in PCR. Figure 2 showstitrations of the final enzyme fraction for both an Ml3template extension of the universal primer (A) and aPCR amplification of a single copy sequence from hu-man DNA (B). Parallel titrations of a less concentratedcommercial preparation of Tuq DNA polymerase areshown for comparison. The Ml3 reactions show a slighttendency to produce shorter primer extension productswhen a vast excess of enzyme is used, but it is the PCRreactions that show the narrowest permissible range ofenzyme concentration. As DNA polymerase concentra-tion is increased as little as fourfold past the optimum, asmear of products first accumulates encompassing bothhigh and low molecular weight species. When the en-zyme concentration is increased still further the prod-uct becomes exclusively an intense band of very smallmolecular weight that has migrated off the bottom ofthe gel in Fig. 2B. This behavior appears to be native tothe Tuq DNA polymerase, since it has been observed inenzyme preparations from varied purification schemesand is equally stable to incubation at 94°C (data notshown). Once a pooled and dialyzed preparation hasbeen titrated, however, the stored aliquots appear toperform consistently at the determined dilution indefi-nitely.ACKNOWLEDGMENTSWe thank Dirk Hunt and Norman Pace for advice and the gift ofthe T. aquaticusstrain, Miriam Greenberg, Lin Zhiwu, and PrashantDesai for help with cell growth and transformation, and Jeffrey Lei-den for helpful discussion. This work was supported by NationalScience Foundation Grant DMB-8901559 to D.R.E. and NIH Grants
  5. 5. 400 ENGELKE ET AL.ROl-HL39137 and ROlHL39693 to D.G. D.G. is a Howard HughesMedical Institute investigator.REFERENCES1. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T.,Erlich, H. A., and Arnheim, N. (1985) Science 230,1350-1354.2. Wong, C., Dowling, C. E., Saiki, R. K., Higuchi, R. G., Erlich,H. A., and Kazazian, H. H., Jr. (1987) Nature (London) 330,384-386.3. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J.(1990) PCR Protocols: A Guide to Methods and Applications,Academic Press, San Diego.4. Chien, A., Edgar, D. B., and Trela, J. M. (1976) J. Bacterial. 127,1550-1557.5. Laemmli, U. K. (1970) Nature (London) 227,680-685.6. Sanger, F., and Coulson, A. R. (1978) FEBS Lett. 87,107-110.7. Lawyer, F. C., Stoffel, S., Saiki, R. K., Myambo, K., Drummond,R., and Gelfand, D. H. (1989) J. Biol. &em. 264,6427-6437.8. Mancuso, D. J., Tuley, E. A., Westfield, L. A., Worrall, N. K.,Shelton-Inloes, B. B., Sorace, J. M., Alevy, Y. G., and Sadler, J. E.(1989) J. Biol. Chem. 264, 19,514-19,527.9. Ginsburg, D., Konkle, B.A., Gill, J. C., Montgomery, R. R., Bock-ensted, P. L., Johnson, T. A., and Yang, A. Y. (1989) Proc. Natl.Acad. Sci USA 86,3723-3727.10. Stark, M. J. R. (1987) Gene 61,255-267.11. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) MolecularCloning: A Laboratory Manual, pp. 368-369, Cold Spring HarborLaboratory, New York.