Abstract

C-peptide (CP), connecting the A and B chains in proinsulin, has been considered to possess physiological effects in diabetes. In order to prolong the half-life of CP in vivo, a long acting CP analog [human serum albumin (HSA-CP)] was obtained by direct gene fusion of a single-chain CP to HSA and expressed in host Pichia pastoris GS115. After 72 h of growth on methanol, the recombinant HSA-CP concentration reached a level of 145 mg/L, representing 70% of total proteins in culture supernatant. The recombinant fusion protein was purified by ultra filtration, Q-sepharose fast flow column and Superdex 75 size-exclusion column. It was specifically recognized by the anti-human HSA antibody and CP antibody in Western blotting assay. In vitro, the recombinant HSA-CP can stimulate HEK293 cell proliferation. In Zucker diabetic fatty (ZDF) rats, 12 weeks recombinant HSA-CP treatment could prevent the accumulation of glomerular extracellular matrix, which leads to mesangial expansion and glomerular hypertrophy. The terminal biological half-time of the protein was 3.38±1.87 h after single administration of 500 nmol/kg of HSA-CP in Wister rats. The pharmacokinetic analysis of the protein indicate its promising application in clinical medicine.

Key words: C-peptide, human serum albumin, recombinant fusion protein, Pichia pastoris, bioactivity, biological half-time.

Abbreviation

CP, C-peptide; HSA-CP, human serum albumin -CP; ZDF, Zucker diabetic fatty;  KW/BW, kidney weight/body weight; GBMT, glomerular basement membrane thickness; FCS, fetal calf serum; SRB, sulphorhodamine B; IRMA, immunoradiometric assay; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; LB, lysogeny broth; PCR, polymerase chain reaction; V(glom), glomerular volume; S(ECM)/S(glom), extracellular matrix area fraction of glomerular cross-section.