基因工程人胰岛素原和胰岛素的分离纯化及性质研究

STUDIES ON GENETIC ENGINEERING OF HUMAN INSULIN——PURIFICATION AND CHARACTERIZATION OF HUMAN PROINSULIN AND INSULIN

携带pWR 590-BCA4质粒的大肠杆菌经发酵培养,提取出人胰岛素原融合蛋白。该融合蛋白经BrCN降解,氧化磺化及QAE-Sepha-dex A-25和Sephadex G-50分离所得到的磺化胰岛素原,经折叠和分离得到了人胰岛素原(HPI)。HPI 再经酶切转化为人胰岛索(HI)和C-肽,HI 的得率为5 m8/L,并获得了HI 的晶体。HPI 和HI 的氨基酸组成、N-端顺序和C-末端分析均与预期值相符。HI 的RIA 活性为猪胰岛素的99%,整体活性为26～27U/mg。

E.coli DH 5 α cells harboring a plas-mid pWR 590-BCA 4 for fused humanproinsulin production were cultured.Thefused human proinsulin was isolated fromthe fermented cells and then subjected it tocleavage with BrCN.The cleaved productwas then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis.Theisolation of human proinsulin-S-sulfonatewas accomplished by ion exchange chroma-tography on QAE-sephadex A-25,followedby gel filtration on sephadex G-50.Thepurified human proinsulin-S-sulfonate wasfolded using a disulfide interchange method.The folding mixture was then chromatogra-phed on sephadex G-50 and purified pro-insulin was obtained.The proinsulin wasthen converted to human insulin and C-peptide by a combination cleavage withtrypsin and carboxypeptidase B.The totalyield of human insulin was about 5 mg/LThe Zinc insulin crystals were obtainedwith amorphous human insulin usingcitrate method.The amino acid compositionN-terminal sequences as well as C-terminalamino acid residues are in agreement withexpected results.The hypoglycemic activityof purified human insulin is 26—27 U/mg,as judged by mouseconvulsion assay,andthe RIA activity is about 99% of that ofprocine insulin.