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Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus
1Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
Pfu, the DNA polymerase from Pyrococcus furiosus, has the lowest error rate of any known polymerase in polymerase chain reaction (PCR) amplification. Previously the protein has been purified from P. furiosus bacterial cultures, and a recombinant form has been produced in a baculovirus system. We have produced a pET plasmid for expression of Pfu in Escherichia coli (the expression plasmid pETpfu is available from ATCC, Accession No. 87496) and found that this plasmid is toxic or unstable in the expressing strain BL21(DE3), even in the absence of induction. However, the plasmid was stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and a 90-kDa protein was expressed upon addition of isopropyl beta-D-thiogalactopyranoside. The protein was purified by heating (to denature E. coli proteins), followed by chromatography on P11 phosphocellulose and mono Q columns. 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.
Insights
Researchers developed a new method for producing Pfu DNA polymerase in Escherichia coli, achieving high purity and activity. This bacterial expression system offers a superior alternative for generating this crucial enzyme for PCR applications.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Pfu DNA polymerase from Pyrococcus furiosus exhibits the lowest error rate for polymerase chain reaction (PCR) amplification.
- Previous production methods included purification from P. furiosus cultures and recombinant expression in a baculovirus system.
Purpose of the Study:
- To develop a stable and efficient bacterial expression system for Pfu DNA polymerase in Escherichia coli.
- To produce a highly active and pure recombinant Pfu DNA polymerase using a novel expression plasmid.
Main Methods:
- A pET plasmid (pETpfu) was engineered for Pfu expression in E. coli.
- The plasmid's stability and toxicity were assessed in BL21(DE3) strains, with and without a pLysS plasmid.
- Protein purification involved heat treatment, P11 phosphocellulose, and mono Q column chromatography.
Main Results:
- The pETpfu plasmid was unstable in BL21(DE3) but stable when co-transformed with the pLysS plasmid.
- A 90-kDa Pfu protein was successfully expressed upon induction.
- The purified bacterial recombinant Pfu showed comparable DNA polymerase and PCR activity to the baculovirus-expressed enzyme.
Conclusions:
- A stable and effective bacterial expression system for Pfu DNA polymerase was established using E. coli.
- This method provides a preferred approach for producing active Pfu DNA polymerase.
- The developed system facilitates the large-scale production of high-fidelity Pfu DNA polymerase for molecular biology applications.