Description:
The dna polymerase from pyrococcus furiosus is referred to here as pfu. Pfu is a thermophilic dna polymerase with as integrated 3'-5' exonuclease activity that corrects error correction is thought to be associated with its intrinsic exonuclease activity. The error rate for pfu is reported to be 7-to 10-fold lower than that of nonproofreading taq dna polymerase, and 2-to 30-fold lower than other proofreading enzymes.
Due to the high fidelity of pfu dna polymerase, it has been widely used in molecular biological research and clinical diagnosis, such as routine pcr, dna sequencing, gene amplification and mutagenesis.

The coding region of pfu gene was riginal amplified from pyrococus furiosus and subcloned into pet expression system. The pet system is the most powerful system yet developed for the cloning and expression of recombinant proteins in e. Coli. Based on the t7 promoter-driven system. Expression of pfu gene was under control of strong bacteriophage t7 transcription and translation signals and expression is induced by providing a source of t7 rna polymerase in the host cell. I. E bl21(de3).

The protein was purified by heating(to denature e. Coli proteins), followed by chromatography on mono q column. The purified protein had the same activity as the commercially obtained baculovirus-expressed pfu in both dna polymerase and pcr reactions. This bacterial expression system appears to be the method of choice for production of pfu today.

The enzyme catalyzes the incorporation of nucleotides into duplex dna in the 5'=>3' direction in the presence of mg2+ at 70-80 cent. Pfu dna polymerase exhibits 3'=>5' exonuclease(proofreading) activity, but has no detectable 5'=>3' exonuclease activity.

Unit definition:
One unit of enzyme catalyzes the incorporation of 10 nanomoles of deoxyribonucleotides into a polynucleotide fraction(adsorbed on de-81) in 30 min at 72 cent.

Activity assay:
20mm tris-hc1(ph8.8 at 25 cent),2.0mm mgso4,10mm(nh4)2so4,10mm kc1,0.1% triton x -100,0.1mg/ml bsa,0.75mm activated calf thymus dna,0.2mm of each dntp,0.4mbq/ml [3h]-dttp.

Storage buffer:
20mm tris-hc1(ph8.2),1mm dtt,0.1mm edta,100mm kc1,0.1% nonidet p40,0.1% tween 20 and 50% glycerol.
10x pcr buffer with mgso4
200mm tris-hc1(ph 8.8 at 25 cent),100mm(nh4)2so4100mm kc1,1% triton x-100,1mg/ml bsa,20mm mgso4.

The pfu dna polymerase expression system contains the following components:
1) pfu expression e. Coli clone strain
2) pfu expression plasmid pet-pfu(20ug)3) operation manual

Host cell: Bl21(de3):
Expression plasmid: Pet11 vector
Selection marker: Amp
Pfu gene size:2328bp
Yield:1x105 units/L