Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed inEscherichia colias inclusion bodies
作者:
Hong Jin,
Mohammad S. Uddin,
Yu L. Huang,
Wah K. Teo,
期刊:
Journal of Chemical Technology&Biotechnology
(WILEY Available online 1994)
卷期:
Volume 59,
issue 1
页码: 67-72
ISSN:0268-2575
年代: 1994
DOI:10.1002/jctb.280590111
出版商: John Wiley&Sons, Ltd.
关键词: rh TNF‐β;E. coli;purification;refolding;inclusion bodies
数据来源: WILEY
摘要:
AbstractHigh level expression of recombinant human tumour necrosis factor β (rh TNF‐β) inEscherichia coliresults 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 rh TNF‐β 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−3alkaline 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 filtratio
点击下载:
PDF
(522KB)
返 回