Jin H, Uddin M S, Huang Y L, Teo W K
Department of Chemical Engineering, National University of Singapore.
J Chem Technol Biotechnol. 1994 Jan;59(1):67-72. doi: 10.1002/jctb.280590111.
High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.
重组人肿瘤坏死因子β（rhTNF-β）在大肠杆菌中的高水平表达导致形成两部分蛋白质，即可溶性活性蛋白和不溶性蛋白，后者无活性并以包涵体（IBs）的形式聚集。在本研究中，设计了一种从包涵体中纯化和复性rhTNF-β的方法，并通过实验验证，产品纯度超过90%，回收率约为30%。该方法包括用特定洗涤缓冲液（Triton X-100/EDTA/溶菌酶/PMSF）洗涤包涵体，用8 mol dm-3碱性尿素溶解包涵体，用离子交换柱纯化，用复性缓冲液复性，最后用超滤膜浓缩和脱盐。通过（1）SDS-PAGE、（2）对小鼠L929细胞的细胞毒性活性、（3）N端氨基酸序列和（4）凝胶过滤色谱法证明，复性蛋白的特性与可溶性部分的纯化蛋白相同。
