PMID:9224900
| Citation |
Chong, S, Mersha, FB, Comb, DG, Scott, ME, Landry, D, Vence, LM, Perler, FB, Benner, J, Kucera, RB, Hirvonen, CA, Pelletier, JJ, Paulus, H and Xu, MQ (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192:271-81 |
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| Abstract |
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), beta-mercaptoethanol (beta-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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| Keywords |
Affinity Labels; Amino Acid Sequence; Base Sequence; Carrier Proteins/chemistry; Chitin; Genetic Vectors; Hydrogen-Ion Concentration; Maltose-Binding Proteins; Methods; Molecular Sequence Data; Protein Processing, Post-Translational; Protein Splicing; Proton-Translocating ATPases/metabolism; Recombinant Proteins/isolation & purification; Temperature; Vacuolar Proton-Translocating ATPases |
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