Shin N K, Kim D Y, Shin C S, Hong M S, Lee J, Shin H C
Laboratory of Protein Engineering, Hanhyo Institute of Technology, Yusong-ku, Taejon, South Korea.
J Biotechnol. 1998 Jun 30;62(2):143-51. doi: 10.1016/s0168-1656(98)00054-6.
Procedures have been devised for producing in Escherichia coli high yields of purified recombinant human growth hormone (hGH), by utilizing N-terminal pentapeptide sequence of human tumor necrosis factor-alpha, histidine tag and enterokinase cleavage site as a fusion partner. The fusion protein was produced as a soluble protein at the beginning of gene expression, but progressively became insoluble in Escherichia coli cytoplasm. The insoluble protein was solubilized by simple alkaline pH shift and purified to near homogeneity by Ni(2+)-chelated affinity chromatography. Following specific enterokinase cleavage, the recombinant hGH was purified by one-step anion exchange chromatography. The ease and speed of this recombinant process, as well as the high productivity, makes it adaptable to the large-scale production of hGH. Moreover, the highly efficient fusion partner could be applied to the production of other therapeutically important proteins.
已设计出相关程序，通过利用人肿瘤坏死因子-α的N端五肽序列、组氨酸标签和肠激酶切割位点作为融合伴侣，在大肠杆菌中高产纯化重组人生长激素（hGH）。融合蛋白在基因表达开始时以可溶性蛋白形式产生，但在大肠杆菌细胞质中逐渐变得不溶。不溶性蛋白通过简单的碱性pH转换进行溶解，并通过镍（2+）螯合亲和层析纯化至接近均一。经过特定的肠激酶切割后，重组hGH通过一步阴离子交换层析进行纯化。该重组过程的简便性、速度以及高生产率使其适用于hGH的大规模生产。此外，这种高效的融合伴侣可应用于其他具有治疗重要性的蛋白质的生产。
