Expression and Physicochemical Characterization of Human Proliferating Cell Nuclear Antigen

Peng Zhang, Shan Jian Zhang, Zhongjian Zhang, J. Frederick Woessner, Marietta Y.W.T. Lee

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41 Scopus citations

Abstract

Human proliferating cell nuclear antigen (PCNA) was overexpressed in Escherichia coli as a soluble protein. Recombinant PCNA was purified to homogeneity by phosphocellulose, Q-Sepharose, Sephacryl S-200, and hydroxylapatite chromatography. Approximately 20 mg of PCNA was isolated from E. coli cells derived from 2 L of culture. Characterization of the recombinant protein showed that it was functionally active and that its properties were similar to those of purified human placental PCNA. Recombinant PCNA stimulated human DNA polymerase δ activity at least 25-fold with poly(dA)/oligo- (dT) as the template. Recombinant PCNA eluted with a Mr, = 102 000 and a Stokes radius of 37 Å by high-performance gel-permeation chromatography. The sedimentation coefficient determined by glycerol gradient ultracentrifugation was 6.3 S. The molecular weight calculated from the Stokes radius and S value was 96 800. The behavior of PCNA was entirely consistent with its being a trimeric protein. Analytical ultracentrifugation and gel filtration revealed the existence of a dimeric species at low dilution. Cross-linking experiments revealed the presence of PCNA dimers which predominated, as well as a trimeric species. These studies provide biophysical evidence that PCNA is an oligomeric protein which behaves as a trimeric species at high protein concentrations but dissociates to a dimer at low protein concentrations.

Original languageEnglish (US)
Pages (from-to)10703-10712
Number of pages10
JournalBiochemistry
Volume34
Issue number34
DOIs
StatePublished - Aug 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Zhang, P., Zhang, S. J., Zhang, Z., Frederick Woessner, J., & Lee, M. Y. W. T. (1995). Expression and Physicochemical Characterization of Human Proliferating Cell Nuclear Antigen. Biochemistry, 34(34), 10703-10712. https://doi.org/10.1021/bi00034a002