Preparation of recombinant murine tumor necrosis factor-alpha in Escherichia coli: a rapid method to remove tags from fusion proteins by thrombin-cleavage and ion-exchange chromatography.
Protein Expr Purif
; 56(1): 138-44, 2007 Nov.
Article
em En
| MEDLINE
| ID: mdl-17703948
ABSTRACT
A recombinant protein of murine tumor necrosis factor (TNF)-alpha 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.
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Coleções:
01-internacional
Contexto em Saúde:
3_ND
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
/
Fator de Necrose Tumoral alfa
Limite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Ano de publicação:
2007
Tipo de documento:
Article