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.
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.
Tsukamoto
Hiroki
H
Department of Immunology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Fukudome
Kenji
K
Kohara
Jun
J
Nakatake
Hiroshi
H
Kimoto
Masao
M
D000818:Animals; D000917:Antibody Formation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013917:Thrombin / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D013917:Thrombin