Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization.
Recombinant human CRP (rhCRP) was secreted into culture supernatant of Escherichia coli by co-expressing kil gene that has a function to secrete colicin E1 outside the cell. Highly purified 5 g rhCRP was produced from 180 L culture supernatant by affinity chromatography. The purified rhCRP was indistinguishable from the native one with respect to Ca(2+)-dependent binding ability to phosphorylcholine, electrophoretic behavior, N-terminal amino acid analysis, and immunochemical properties. The molecular weight of rhCRP monomer was determined to be 23059.7 Da by TOF/MS analysis. These results indicate that rhCRP has the same protein structure as native one and that rhCRP has the potential as a reference material and/or calibrator of high-sensitivity CRP assay to predict the risk of cardiovascular disease.
Tanaka
Toshio
T
Nagahama Institute for Biochemical Science, Oriental Yeast Co., Ltd., 50 Kano-cho, Nagahama-shi, Shiga 526-0804, Japan.
Horio
Takekazu
T
Matuo
Yuhsi
Y
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002097:C-Reactive Protein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006801:Humans; D007118:Immunoassay; D008970:Molecular Weight; D010767:Phosphorylcholine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C069003:kil protein, E coli; D010767:Phosphorylcholine; D002097:C-Reactive Protein; D002118:Calcium