CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF THE GENE OF THE THERMOSTABLE DNA-POLYMERASE OF THERMUS-AQUATICUS YT1

A piece of the genomic DNA of Thermus aguaticus YT1 containing the thermostable DNA polymerase (Taq polymerase, EC 2.7.7.7) gene was cloned into phasmid vector pSL5. A BglII fragment of this genome piece with the Taq polymerase gene was inserted into the BamH1 site of plasmid pUC19 polylinker sequence. In order to enhance Taq polymerase gene expression in Escherichia coli, it was cloned within the reading frame of the initiation codon ATG of vector pPR-TGATG-1 under the control of promoter P-R and temperature- sensitive repressor of phage lambda and the regulatory sequences of E. coli tryptophan operon. This was accomplished by amplification of a short 5'-terminal fragment of the coding part of the Taq polymerase gene by polymerase chain reaction (PCR) and incorporation into it of an artificial SacI restriction site. The fragment was cloned into SacI-digested vector pPR-TGATG-1, and the lacking part of the natural gene was inserted into the internal KpnI site of the amplified fragment. The cells of the resulting E. coli strain PVG-A1 readily expressed the Taq polymerase gene at a nonpermissive temperature. Recombinant Taq polymerase constituted up to 1-2% of the total bacterial cell protein. When purified to a nearly homogeneous state, it amplified DNA fragments of up to 5,500 bp in PCR and could be be used for DNA sequencing according to Sanger. The purified Taq polymerase with a specific activity of 180,000-200,000 un./mg was inactivated with a half-time of 60 min at 95 degrees C, and retained its activity for at least 65 conventional PCR cycles. The recombinant E. coli PVG-A1 strain thus produced allows one to obtain up to 500,000 units of purified recombinant Taq polymerase from 2 liters of bacterial culture.