Expression and physicochemical characterization of human proliferating cell nuclear antigen.
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.
Zhang
P
P
Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101, USA.
Zhang
S J
SJ
Zhang
Z
Z
Woessner
J F
JF
Lee
M Y
MY
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004258:DNA Polymerase III; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000235:genetics; D018398:Homeodomain Proteins; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D015778:Minor Histocompatibility Antigens; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010920:Placenta / Q000737:chemistry; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014461:Ultracentrifugation
C096816:BCL2-related protein A1; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D051818:Replication Protein C; D008274:Magnesium