Overexpression and rapid purification of biologically active yeast proliferating cell nuclear antigen.
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 delta in vitro. We have expressed yPCNA to a high level (> or = 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) delta tag on the polypeptide permitted rapid and high-yield single-step purification of the protein (approximately 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.
Biswas
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EE
Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201, USA.
Chen
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PH
Biswas
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SB
D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000276:immunology*
D017931:DNA Primers; D004271:DNA, Fungal; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins