Preparation of recombinant murine tumor necrosis factor-α in Escherichia coli: A rapid method to remove tags from fusion proteins by thrombin-cleavage and ion-exchange chromatography

Hiroki Tsukamoto, Kenji Fukudome, Jun Kohara, Hiroshi Nakatake, Masao Kimoto

研究成果: Article査読

12 被引用数 (Scopus)

抄録

A recombinant protein of murine tumor necrosis factor (TNF)-α was expressed in Escherichia coli (E. coli) by using a pET Trx Fusion System. The fusion protein was effectively solubilized and purified by Ni-affinity chromatography. A high concentration of thrombin quickly and specifically cleaved the introduced site between the tags and the target fragment. We found that thrombin tightly bound to an ion-exchange resin, CM-Sepharose, under conditions avoiding adsorption of most proteins. By passing through the column, thrombin was quickly removed from the reaction mixtures. These methods appear to be widely potentially useful to remove the tags from recombinant fusion proteins. Prepared recombinant TNF demonstrated cytotoxic effects to L929 cells at very low concentrations with an EC50 value of 0.19 ± 0.02 pM. In addition, immunization of a rabbit with the protein induced a neutralizing antibody. The methods used in this study appear to be useful to prepare significant amount of soluble functional recombinant proteins in E. coli.

本文言語English
ページ(範囲)138-144
ページ数7
ジャーナルProtein Expression and Purification
56
1
DOI
出版ステータスPublished - 2007 11 1

ASJC Scopus subject areas

  • Biotechnology

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