C-Peptide (CP) can inhibit diabetic complication, but the half-life of CP is short in vitro. The gene of CP was fused with gene of Human serum albumin (HSA) to extend half-life of CP; the fusion protein named human serum albumin- C-Peptide fusion protein (HSA-CP) was expressed in recombinant Pichia pastoris. When Pichia pastoris/HSA-CP was cultivated in mineral Sal- medium, it produced small amount of HSA-CP. The desired protein was found partially degraded by the proteolytic activity in the broth; cause the loss of the final recovery of HAS-CP.The major results of this study were presented as follow:The key factors on high-level HSA-CP production in Pichia pastoris/HSA-CP were investigated in mineral Sal-medium in shake flask. The optimal initial glycerol concentration was 20g/L, and dry cell weight reached 10.64g/L; the yield of HSA-CP reached 42.97 mg/L with 10g/L methanol induction for 72h. The yield of HSA-CP reached 368.45mg/L in the low cell density culture with mineral Sal- medium in 7L fermenter, dry cell weight was 56.43 g/L; when high cell density cultured, the yield of HSA-CP reached 707.3mg/L, dry cell weight was143.09 g/L.The methods to slow down the proteolytic degradation of HSA-CP were researched. Protease could decrease HSA-CP into peptide fragment. As a result, it was found that adding (NH4)2SO4 had little effect on HSA-CP degradation, adding casamino acid could slow down the degradation of HSA-CP. In the induction phase, adding 1.5% casamino acid and keep temperature at 26℃, protease activity reduced from 330.9 U/ml to 231.4 U/ml in shake flask, and from 511.4 U/ml to 442.2 U/ml in low cell density cultivation, but it has little effect on high cell density cultivation.The major steps of HSA-CP separation and purification were determined. Different concentrate methods were investigated, the result was: 7.04g/L HSA-CP was obtained by ultra-filtration, to get 20 folds concentration, the recovery rate was 82.8%. After DEAE Sepharose F.F. running, the HSA-CP was shown as a single band through SDS-PAGE electrophoresis. HSA-CP extracted was specifically immune-reacted with an anti-human HSA polyclonal antibody and CP polyclonal antibody in Western blot assay.
|