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High-Level Expression, Purification, and Thermus Aquatlcus DNA

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High-Level Expression, Purification, and Thermus Aquatlcus DNA Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press High-level Expression, Purification, and Enzymatic Characterization of Full-length Thermus aquatlcus DNA Polymerase and a Truncated Form Deficient in 5' to 3' Exonuclease Activity Frances C. Lawyer, 1 Susanne Stoffel, 1 Randall K. Saiki, 2 Sheng-Yung Chang, 3 Phoebe A. Landre, ~ Richard D. Abrarnson, 1 and David H. Gelfand 1 1Program in Core Research and Departments of 2Human Genetics and 3Infectious Disease, Roche Molecular Systems, Alameda, California 94501 The Thermus aquaticus DNA poly- life of 9 min at 97.5~ The Stoffel I in the native host is quite low (0.01- merase I (Taq Pol I) gene was cloned fragment has a half-life of 21 min at 0.02% of total protein). The cloning and into a plasmid expression vector that 97.5~ Taq Pol I contains a polymer- expression of full-length 94-kD Taq Pol I utilizes the strong bacteriophage ization-dependent 5' to 3' exonu- in E. coli under control of the E. coli lac PL promoter. A truncated form of Taq clease activity whereas the Stoffel promoter r or the tac promoter (7~ has Pol I was also constructed. The two fragment, deleted for the 5' to 3' ex- been reported. Because polymerase constructs made it possible to com- onuclease domain, does not possess yields in these constructs were low pare the full-length 832-amino-acid that activity. A comparison is made (-0.01% of total protein in our initial Taq Pol I and a deletion derivative among thermostable DNA poly- construct; see ref. 5), we sought to im- encoding a 544-amino-acid transla- merases that have been character- prove the expression level of the enzyme tion product, the Stoffel fragment. ized; specific activities of 292,000 by mutagenizing the 5' and 3' ends of Upon heat induction, the 832-amino- units/mg for Taq Pol I and 369,000 the gene and cloning the mutagenized acid construct produced 1-2% of to- units/mg for the Stoffel fragment are gene into a more suitable expression vec- tal protein as Taq Pol I. The induced the highest reported. tor. We also constructed a truncated Taq 544-amino-acid construct produced Pol I gene, deleted for the first 867 bp of 3% of total protein as Stoffel frag- the gene, which yields a predicted 61-kD ment. Enzyme purification included translation product. The 61-kD deriva- cell lysis, heat treatment followed by tive, Taq Pol I Stoffel fragment, is active Polymin P precipitation of nucleic ac- The thermostable DNA polymerase I in polymerase assays and PCR and is de- ids, phenyl sepharose column chro- (Taq Pol I) from Thermus aquaticus (Taq) void of the inherent 5' to 3' exonuclease matography, and heparin-Sepharose greatly improves the yield, specificity, activity of Taq Pol I. A similarly trun- column chromatography. For full- automation, and utility of the poly- cated gene deleted for the first 705 bp, length 94-kD Taq Pol I, yield was merase chain reaction (PCR) ~l'z~ method has recently been constructed, yielding 3.26x 107 units of activity from 165 of amplifying DNA fragments. ~3~ Fur- an approximately 67-kD translation grams wet weight cell paste. For the thermore, because of the high turnover product, KlenTaq DNA polymerase. (8~ 61-kD Taq Pol I Stoffel fragment, the number, lack of proofreading activity, Protein produced by the two constructs yield was 1.03 • 106 units of activity high temperature optimum, and ability described here provides a plentiful sup- from 15.6 grams wet weight cell to incorporate 7-deaza-2-deoxyguano- ply of the two forms of Taq Pol I enzyme, paste. The two enzymes have maxi- sine efficiently, Taq Pol I yields long thereby enabling their biochemical mal activity at 75~ to 80~ 2-4 mM stretches of readable DNA sequence that properties to be investigated. The study MgClz and 10-55 mM KCI. The nature are uniform in intensity and free of of these enzymes not only provides key of the substrate determines the pre- background. ~4~ insights into nucleic acid metabolism, cise conditions for maximal enzyme A 94-kD Taq Pol I has been purified but also provides useful information to activity. For both proteins, MgCI2 is from Taq, but growing the organism is those investigators who exploit DNA the preferred cofactor compared to more difficult than growing Escherichia polymerases in specialized molecular bi- MnCI z, CoCIz, and NiCI 2. The full- coll. Although the activity yield is high ology techniques, including the PCR. length Taq Pol I has an activity half- (40--60%), the expression level of Taq Pol Advancements in PCR methodology 2:275-2879 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $3.00 PCR Methods and Applications 275 Downloaded from genome.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press depend in part on an increased under- (pH 7.2), 5 mM MgC12.(17~ Single- tion products were transformed into standing of the biochemistry of thermo- stranded template DNA was prepared by DG98. Nitrocellulose filters containing stable DNA polymerases, as such conventional methods r from M13 ampicillin-resistant transformants were information aids substantially in the phage TSYC657 (7.359 kb), a derivative hybridized according to the method of constant refinement of this and other re- of M13mpl0 containing a 129-nucle- Woods et al. (23) with 3zp-labeled DG26 search techniques. otide insert derived from a Streptornyces probe. Positive colonies were picked and limosus a-amylase gene. (~8~ The 24-mer screened by restriction analysis of alka- synthetic oligodeoxynucleotide primer line-SDS miniptasmid preparations. A MATERIALS AND METHODS SC64 (Table 1) is complementary to the representative correct candidate was des- Bacterial Strains and Plasmids S. limosus-derived insert in TSYC657 and ignated pLSG2 (Fig. 1). has a calculated melting temperature of For mutagenesis of pLSG2, 0.5 pmole E. coli strain DG98 (thi-1 endA1 hsdR17 95~ at 0.12 I~M in 100 mM NaCI. Primer of single-stranded pLSG2, 0.5 pmole of lacI Q IacZAM15 proC::TnlO supE44/ F'la- and template were annealed at $83 nM PvuII-digested, CIAP-treated pBS § and cIQ IacZAM15 proC +) has been de- SC64 and 256 nM TSYC657(2.2:l primer: 2.5 pmoles of oligonucleotide SC107 scribed. (9~ E. coli strain DGl16 [thi-1 template) in 10 mM Tris-HC1 (pH 8.0) 6 (Table 1) were annealed. Extension, endA1 hsdR17 supE44 (hcI857 bloT76 mM MgCl2, and 50 mM KCI. The mixture transformation, and screening were per- M-/1)] is a derivative of strain MM294 (1~ was heated at 95~ for 4 rain, incubated formed as described above, using 32p-la- containing a defective h prophage. at 70~ for 10 min, and cooled to room beled SC107 as the probe. A representa- DGl16 was prepared using bacterioph- temperature. tive correct candidate was designated age P1 transduction ~1~ and E. coli strain pSYC1578 (Fig. 1). N6590 [C600 rK- mK- thr leu pro IacZXA21::Tn10 (hcI857 bloT76 M-/1), Cloning Procedures Assembly of a Full-length Taq Pol I provided by M. Gottesman, Columbia For oligonucleotide site-directed mu- Gene in High-level Expression University] as the source of the defective tagenesis, single-stranded DNA was pre- Vectors prophage. Plasmid pBS § was purchased pared from pBS § plasmid derivatives from Stratagene. Plasmid pFC54.T has pLSG1 and pLSG2 as described. (s~ Com- An SphI and BglII-digest of pSYC1578 been described. (~z~ Plasmid pDG160, a petent cells were prepared and transfor- DNA was ligated with HindIII- and derivative of pFC54.T, contains a conve- mations were carried out by the method BamHI-digested vector pFC54.T and an nient restriction site polylinker between of Hanahan. (19~ Alkaline-SDS miniplas- annealed duplex oligonucleotide, DG271 the PL promoter and retroregulator cas- mid DNA preparations were carried out 28 (Table 1). The oligonucleotide duplex settes. by the method of Birnboim and Doly. (2~ provided HindIII and SphI cohesive ends Enzymatic dideoxynucleotide DNA se- and codons one, two, and two of three Reagents quence analysis r confirmed all DNA nucleotides for codon three of Taq Pol I. sequence alterations. The resulting plasmid was designated Restriction endonucleases [New England pLSG5 (Fig. 1). Biolabs (NEB)], E. coli DNA polymerase I, Oligonucleotide Site-directed large fragment (Klenow) (U.S. Biochem- Mutagenesis (22) Assembly of a Truncated Taq Pol I icals), T4 DNA ligase, (~3~ calf intestine al- Gene kaline phosphatase (CIAP) (Boehringer- For pLSG1 mutagenesis, 0.24 pmole of Mannheim), and polynucleotide kinase single-stranded pLSG1, 0.36 pmole of A BstXI digest of pSYC1578 was treated (kinase) (NEB) were used according to PvuII-digested CIAP-treated vector pBS +, with Klenow to generate blunt ends. The standard procedures. Oligonucleotides and 0.48 pmole of primer DG26 (Table DNA was then treated with BglII and a were phosphorylated as described. (~4~ 1) were annealed. The mixture was put 1619-bp BstXI-blunt-BgllI fragment (en- Molecular weight standards for SDS- on ice and TTP, dGTP, dATP, [3H]dCTP coding Taq Pol I codons 294-832) was polyacrylamide gel electrophoresis were each added to 200 p.M along with 1 isolated via gel electrophoresis and elec- (PAGE) were purchased from Pharma- unit of Klenow fragment. The extension troelution. The isolated DNA fragment cia [~/-3zp]ATP (3000 Ci/mmole), mixture was incubated at 0~ for 30 min was ligated to HindlII- and BamHI-di- [R-32p]dCTP (800 Ci/mmole), and and then at 30~ for 30 rain. The reac- gested vector pFC54.T and an annealed [3H]dCTP (22.8 Ci/mmole), were pur- chased from New England Nuclear.
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