Wei G, Tang J G
Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, China.
Biochem Mol Biol Int. 1995 Nov;37(5):895-901.
Des-B30 single-chain insulin gene was constructed from chemically synthesized DNA encoding human proinsulin by oligo-nucleotide induced deletion, cloned into the expression plasmid pBV220, and expressed in E. coli with a level of around 5%. The expressed product was in the form of inclusion bodies. After downstream processing, 45 mg of des-B30 single-chain insulin with a purity of up to 90% was obtained from 1 liter of high density fermentation medium. It is suggested that the fusion protein model to increase the stability of small proteins seems inadequate to some extent, the formation of inclusion bodies may be the key factor for the stability of some expressed products, large or small, in the cytoplasm of E. coli cells.
通过寡核苷酸诱导缺失从化学合成的编码人胰岛素原的DNA构建去B30单链胰岛素基因，将其克隆到表达质粒pBV220中，并在大肠杆菌中表达，表达水平约为5%。表达产物以包涵体形式存在。经过下游处理，从1升高密度发酵培养基中获得了45毫克纯度高达90%的去B30单链胰岛素。这表明增加小蛋白质稳定性的融合蛋白模型在一定程度上似乎并不充分，包涵体的形成可能是大肠杆菌细胞质中某些大小表达产物稳定性的关键因素。
