The preparation of recombinant rainbow trout insulin like growth factorsⅠ(IGF Ⅰ) andⅡ(IGF Ⅱ) using the His6 fusion polypeptide technique and the mitogen ic activities of these compounds were investigated. Rainbow ...The preparation of recombinant rainbow trout insulin like growth factorsⅠ(IGF Ⅰ) andⅡ(IGF Ⅱ) using the His6 fusion polypeptide technique and the mitogen ic activities of these compounds were investigated. Rainbow trout IGF Ⅰand IGF ⅡB C A D domain cDNA were subcloned into expression vector pET 15b (Nova gen, USA) and expressed in host E. coli BL21 (DE3) as fusion polypeptides co ntai ning a stretch of 6 histidines at the N terminus (referred as His6 fusion polyp e ptide). The addition of IPTG (0 4 mmol/L) induced the expression of polypeptide s with a molecular weight of about 10 0 kDa. The expressed proteins were ammoniu m sulfate fractionally precipitated and further purified using a Ni 2+ His ·Bind R esin (Novagen, USA) affinity column. The reduced SDS PAGE showed that IGF Ⅰ w as of 80% purity and IGF Ⅱ was of 90% purity. The rainbow trout IGF Ⅰ His6 fusi on polypeptide showed dose dependent mitogenic effects on BALB/NIH3T3 cells in th e serum free medium culture, and rainbow trout IGF Ⅱ His6 fusion polypeptide al s o showed this kind of activity, but with lower potency. Taken together,these re sults indicated that the rainbow trout IGF Ⅰ and ⅡHis6 fusion polypeptides pr o duced in E. coli were biologically active, with IGF Ⅰ His6 fusion polypept ide of higher potency.展开更多
文摘The preparation of recombinant rainbow trout insulin like growth factorsⅠ(IGF Ⅰ) andⅡ(IGF Ⅱ) using the His6 fusion polypeptide technique and the mitogen ic activities of these compounds were investigated. Rainbow trout IGF Ⅰand IGF ⅡB C A D domain cDNA were subcloned into expression vector pET 15b (Nova gen, USA) and expressed in host E. coli BL21 (DE3) as fusion polypeptides co ntai ning a stretch of 6 histidines at the N terminus (referred as His6 fusion polyp e ptide). The addition of IPTG (0 4 mmol/L) induced the expression of polypeptide s with a molecular weight of about 10 0 kDa. The expressed proteins were ammoniu m sulfate fractionally precipitated and further purified using a Ni 2+ His ·Bind R esin (Novagen, USA) affinity column. The reduced SDS PAGE showed that IGF Ⅰ w as of 80% purity and IGF Ⅱ was of 90% purity. The rainbow trout IGF Ⅰ His6 fusi on polypeptide showed dose dependent mitogenic effects on BALB/NIH3T3 cells in th e serum free medium culture, and rainbow trout IGF Ⅱ His6 fusion polypeptide al s o showed this kind of activity, but with lower potency. Taken together,these re sults indicated that the rainbow trout IGF Ⅰ and ⅡHis6 fusion polypeptides pr o duced in E. coli were biologically active, with IGF Ⅰ His6 fusion polypept ide of higher potency.
