Expression and Physicochemical Characterization of Human Proliferating Cell Nuclear Antigen
| Autor: | J. F. Woessner, Peng Zhang, Marietta Y.W.T. Lee, Shan-Jian Zhang, Zhongjian Zhang |
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| Rok vydání: | 1995 |
| Předmět: |
Models
Molecular Saccharomyces cerevisiae Proteins Low protein Protein Conformation Placenta Molecular Sequence Data Size-exclusion chromatography Gene Expression DNA-Directed DNA Polymerase Biochemistry law.invention Minor Histocompatibility Antigens Replication factor C law Proliferating Cell Nuclear Antigen Humans Magnesium Replication Protein C Polyacrylamide gel electrophoresis DNA Polymerase III Stokes radius Cell Nucleus Homeodomain Proteins Chromatography Base Sequence biology Chemistry Molecular biology Recombinant Proteins Proliferating cell nuclear antigen DNA-Binding Proteins Molecular Weight Repressor Proteins Cross-Linking Reagents Proto-Oncogene Proteins c-bcl-2 Chromatography Gel Recombinant DNA biology.protein Electrophoresis Polyacrylamide Gel Ultracentrifuge Ultracentrifugation |
| Zdroj: | Biochemistry. 34:10703-10712 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00034a002 |
| Popis: | 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 delta activity at least 25-fold with poly(dA)/oligo-(dT) as the template. Recombinant PCNA eluted with a M(r) = 102,000 and a Stokes radius of 37 Angstrum 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. |
| Databáze: | OpenAIRE |
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