کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2021816 1069264 2007 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
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
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
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
چکیده انگلیسی

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 56, Issue 1, November 2007, Pages 138–144
نویسندگان
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