Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli
Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli
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DOI：
10.1006/prep.1999.1179
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发表时间：
2000-03-01
影响因子：
1.6
通讯作者：
Panda, AK
中科院分区：
文献类型：
--
作者：
Patra, AK;Mukhopadhyay, R;Panda, AK
重组人生长激素(r-hGH)在大肠杆菌中以包涵体形式表达。在补料分批发酵10h，细胞浓度为25g干重/L的条件下，r-hGH产量为1.6g/L。用含还原剂和不含还原剂的不同缓冲液从包涵体中增溶r-hGH，并与8M尿素和6M GDN-HCl的增溶程度进行了比较。在大肠杆菌中高效表达时，疏水相互作用和离子相互作用是形成r-hGH包涵体的主要原因。在含2 M尿素的pH为12.5的100 mM Tris缓冲液中，r-hGH包涵体完全增溶。在低浓度尿素存在下，r-hGH包涵体的溶解有助于保留r-hGH原有的天然二级结构，从而提高复性过程中生物活性蛋白的产量。在含有2M尿素的Tris缓冲液中溶解的r-hGH在缓冲液交换过程中不易聚集，因此可以通过简单的稀释复性。用DEAE-Sepharose型离子交换层析对r-hGH进行纯化，并通过尺寸排斥层析得到纯度较高的单体r-hGH。纯化的单体r-hGH的总产率接近于最初包涵体蛋白的50%，并被发现具有促进大鼠NB2淋巴瘤细胞系生长的生物活性。(C)2000年学术出版社。
Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was similar to 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines. (C) 2000 Academic Press.