Overexpression and Rapid Purification of Biologically Active Yeast Proliferating Cell Nuclear Antigen

doi:10.1006/prep.1995.0007

Protein Expression and Purification

Volume 6, Issue 6, December 1995, Pages 763-770

https://doi.org/10.1006/prep.1995.0007 Get rights and content

Abstract

Yeast proliferating cell nuclear antigen (yPCNA) is a cell-cycle-regulated protein that has been shown to be required for the efficient elongation of primed DNA templates by DNA polymerase δin vitro.We have expressed yPCNA to a high level (≥30% of the total cellular protein) with and without a six-residue histidine tag at its amino-terminus. Both forms of the recombinant protein were found to be biologically active and no significant differences were observed between the two forms. In this report we describe an efficient method of extraction of DNA binding proteins, such as yPCNA, overexpressed inEscherichia coli.The presence of a (His)₆tag on the polypeptide permitted rapid and high-yield single-step purification of the protein (∼60 mg of purified yPCNA per liter of induced cell culture) by immobilized metal affinity chromatography using an imidazole gradient elution procedure. The purified yPCNA was used to characterize the biological activity and tertiary structure of the protein. Chemical crosslinking and size-exclusion FPLC studies indicated that both forms of the protein have a trimeric–oligomeric structure in solution. Taken together these results indicate that both forms of the recombinant yPCNA were similar to the endogenous protein in their biochemical properties. The strategies presented here are designed to maximize the yield of recombinant protein and should prove useful to the purification of other recombinant DNA binding proteins.

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Regular ArticleOverexpression and Rapid Purification of Biologically Active Yeast Proliferating Cell Nuclear Antigen

Abstract

Regular Article
Overexpression and Rapid Purification of Biologically Active Yeast Proliferating Cell Nuclear Antigen