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Expression and physicochemical characterization of human proliferating cell nuclear antigen
1Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101, USA.
Biochemistry
|August 29, 1995
Summary
Human proliferating cell nuclear antigen (PCNA) was successfully produced as a soluble, functional protein in E. coli. Biophysical studies demonstrate that PCNA exists as a trimer at high concentrations and dissociates into dimers at lower concentrations.
Area of Science:
- Molecular Biology
- Protein Biochemistry
Background:
- Proliferating cell nuclear antigen (PCNA) is a crucial protein involved in DNA replication and repair.
- Understanding the oligomeric state of PCNA is essential for elucidating its function.
Purpose of the Study:
- To overexpress and purify human PCNA in Escherichia coli.
- To characterize the biophysical properties and oligomeric state of recombinant human PCNA.
Main Methods:
- Recombinant protein expression in E. coli.
- Multiple chromatography techniques for purification (phosphocellulose, Q-Sepharose, Sephacryl S-200, hydroxylapatite).
- High-performance gel-permeation chromatography, glycerol gradient ultracentrifugation, analytical ultracentrifugation, and cross-linking experiments for biophysical characterization.
Main Results:
- Successfully isolated approximately 20 mg of soluble, homogeneous recombinant human PCNA.
- Recombinant PCNA demonstrated functional activity, stimulating human DNA polymerase delta.
- Biophysical analyses indicated PCNA exists as a trimer at high concentrations, dissociating to a dimer at low concentrations.
Conclusions:
- Recombinant human PCNA produced in E. coli is functionally active and exhibits similar properties to native PCNA.
- PCNA is an oligomeric protein that exists in a concentration-dependent equilibrium between trimeric and dimeric forms.