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

Authors
Journal
Protein Expression and Purification
1046-5928
Publisher
Elsevier
Publication Date
Volume
6
Issue
6
Identifiers
DOI: 10.1006/prep.1995.0007
Disciplines
  • Biology
  • Chemistry
  • Design
  • Medicine

Abstract

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 δ in 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 in Escherichia 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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