Purification of Proteins Fused to Either the Amino or Carboxy Terminus of the Mycobacterium xenopi Gyrase A Intein
| Autor: | Maurice W. Southworth, Kensey Amaya, Thomas C. Evans, Ming-Qun Xu, Francine B. Perler |
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| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 27, Iss 1, Pp 110-120 (1999) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/99271st04 |
| Popis: | The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single- splice-junction autocleavage element. When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step. Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16°–25°C. Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M. xenopi gyrase A mini-intein vs. the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACTTM purification system. Furthermore, the M. xenopi gyrase A mini-intein Cterminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis. |
| Databáze: | Directory of Open Access Journals |
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