Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element
1997; Elsevier BV; Volume: 192; Issue: 2 Linguagem: Inglês
10.1016/s0378-1119(97)00105-4
ISSN1879-0038
AutoresShaorong Chong, Fana B. Mersha, Donald G. Comb, Melissa E. Scott, David Landry, Luis M. Vence, Francine B. Perler, Jack S. Benner, Rebecca Kucera, Christine A Hirvonen, John Pelletier, Henry Paulus, Ming-Qun Xu,
Tópico(s)RNA and protein synthesis mechanisms
ResumoA 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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