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Updated: Aug 14, 2026
The Multifaceted Benefits of Protein Co-expression in Escherichia coli
Published on: February 5, 2015
Coexpression of the subunits of T7 DNA polymerase from an artificial operon allows one-step purification of active
Joyce Chiu1, Daniel Tillett, Paul E March
1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Abstract:
T7 DNA polymerase expression was performed from an artificial operon by cloning its cofactor, thioredoxin, downstream of a N-terminal 9xHis-tagged T7 gene 5 (gp5). Up to 90% of gp5 was soluble in the presence, but not in the absence of thioredoxin coexpression suggesting that free-form thioredoxin assisted solubilization of gp5. Expression and single-step nickel-agarose affinity purification resulted in recovery of an enzyme that was 97% pure. Copurification of thioredoxin was observed and the estimated molar ratio of thioredoxin to gp5 was 1:1 in the purified DNA polymerase complex. Purified T7 DNA polymerase exhibited full polymerase activity compared to the commercial enzyme and required no exogenous thioredoxin for activity.
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