Purification and characterization of Cop, a protein involved in the copy number control of plasmid pE194.
Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl. Cop protein was calculated to contain 39.1% alpha-helix, 16.8% beta-sheet, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.
Kwak
J H
JH
School of Bioscience and Food Technology, Handong University, Pohang, Korea.
Kim
J
J
Kim
M Y
MY
Choi
E C
EC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004269:DNA, Bacterial / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; C065826:cop protein, Bacteria