Enzymes, Primers, and Cloning
Restriction endonucleases and enzymes used in cloning DNA were obtained from Promega Biotech (Madison, WI, USA). Oligonucleotides used as primers were produced by DNA International (Lake Oswego, OR, USA). Preparation and cloning of DNA fragments and vectors was performed by standard techniques (1).
Construction of the Expression Vector
The cloning of the pTAQ expression vector is similar to that described previously (4) with the use of the more common pUC18 vector in place of the no longer available pTTQ18 expression vector (Amersham Corp., Arlington Heights, IL, USA). Briefly, genomic DNA was isolated from T. aquaticus and used as a template to amplify the Taq DNA polymerase gene by Polymerase Chain Reaction (PCR) (4). The amino terminal primer was 5'-CACGAATTCGGGGATGCTGCC-CCTCTTTGAGCCCAAG, creating a unique underlined EcoRI restriction cut site. The carboxyl terminal primer was 5'-GTGAGATCTATCACTCCTTGGCGGAGAGCCAGTC, creating the unique underlined BglII restriction cut site. The resulting amplification product was digested with EcoRI and BglII and cloned into pUC18 digested with EcoRI and BamHI. This resulted in a fusion that provides the initiator methionine of the vector, replacing the first two amino acids encoded by the native enzyme (Met-Arg-...) with Met-Asn-Ser-...(Gene Bank Accession No. J04639). This vector was designated pTAQ and transformed into DH5a by Hanahan's method for bacterial transformation (5).
Purification of DNA Polymerase
Taq DNA polymerase was purified from 100 mL cultures by a simplification of a previous method (4,7,10). Super Broth media (1) plus ampicillin was inoculated with 100 uL of a log phase culture of DH5a containing the pTAQ construct described above. This culture was grown to an OD600 of 0.3 at 37 oC, then induced with 0.5 mM IPTG and allowed to grow for 16 hours. An aliquot was saved at -20 oC for protein and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. An aliquot was taken and sonicated with a Branson Sonifier 250 (Branson Unltrasonics Corporation, Danbury, CT, USA) and assayed for activity either on the same day or stored at -20 oC in 50% glycerol for assay at a later time. Cells were centrifuged, resuspended in three mL of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA) containing 4 mg/mL lysozyme (Sigma Chemical Co., St. Louis, MO, USA), and incubated 15 minutes at room temperature. Three mL of buffer B was added (10 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM EDTA, 0.5% Tween 20, 0.5% Nonidet P40), and the mixture was incubated 60 minutes at 75 oC in a shaking water bath. Cell debris and denatured protein were removed by centrifugation at 12,000 x g for 10 minutes at 4 oC in a Sorvall RC2B high speed centrifuge using an SS34 rotor. This lysate was then mixed with an equal volume of storage buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 1% Triton X-100) containing 50% glycerol, followed by addition of an equal volume of storage buffer containing 75% glycerol bringing the final glycerol concentration to 50% and diluting the lysate 4 times. This mixture was then stored at -20 oC.
Protein and Activity Assays
Protein levels were determined using the procedure and supplies of BioRad Laboratories (Hercules, CA, USA) based on the method of Bradford (2). Absorbance at 595nm was measured using a Pharmacia Ultrospec III (Pharmacia LKB Biochrom Ltd., Cambridge, England). SDS-PAGE (6) was performed as described in Figure 1. Enzymatic activity of thermostable DNA polymerase was analyzed by PCR titrations and compared to commercial Taq enzyme (Promega Biotech, Madison, WI, USA). Induced cells were also tested for polymerase activity. The cells were lysed open by sonication as described previously, diluted up to 1:1600, and one uL aliquots from these dilutions were then added to 20uL of PCR reaction mixture and cycled as described in Figure 2. Five uL of this PCR reaction mix was then mixed with two uL of loading buffer and loaded onto a 1% agarose gel and electrophoresed (1).