Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei.
Complete PCR-derived DNA fragments containing the structural genes for DNA polymerases of the archaeons Pyrococcus furiosus and Pyrococcus woesei were cloned into an expression vector. The clones expressing thermostable His-tagged DNA polymerases were selected. The cloned fragments were sequenced. The DNA sequences were verified to be authentic by sequencing several clones. The nucleotide (nt) sequence revealed that DNA polymerase of P. woesei (Pwo DNA polymerase) consists of 775 amino acids and has a molecular weight of 90,566. It shows 100% nucleotide identity to the nucleotide sequence of DNA polymerase from P. furiosus (Pfu DNA polymerase). The results confirm that nucleotide sequences of both archaeons (P. furiosus and P. woesei) are highly similar. The recombinant DNA polymerases (His-tagged Pfu and His-tagged Pwo) contained a polyhistidine tag at the N-terminus (43 additional amino acids) that allowed single-step isolation by Ni-affinity chromatography. We found that recombinant plasmids are toxic or unstable in the expressing strain BL21(DE3), even in the absence of the inducing agent, IPTG. However, the plasmids were stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were expressed upon IPTG addition. The proteins were purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni2+-Sepharose columns. The enzymes were characterized and displayed high DNA polymerase activity and thermostability. This bacterial expression system appears to be the method of choice for production of Pfu or Pwo DNA polymerases.
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, Gdańsk, 80-952, Poland.
S
S
Dabrowski
J
J
Kur
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D019766:Genes, Archaeal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
D017931:DNA Primers; D019641:DNA, Archaeal; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase