Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element

来自 Elsevier

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阅读量：

184

作者：

S Chong，FB Mersha，DG Comb，ME Scott，D Landry，LM Vence，FB Perler，J Benner，RB Kucera，CA Hirvonen

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摘要：

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae ( Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), β-mercaptoethanol (β-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

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关键词：

cutaneous afferent nerve fibre nociceptor

DOI：

10.1109/84.825779

被引量：

1415

年份：

1997