Purification of Proteins Fused to Either the Amino or Carboxy Terminus of the Mycobacterium xenopi Gyrase A Intein
| Autor: | Thomas C. Evans, Maurice W. Southworth, Kensey R. Amaya, Ming-Qun Xu, Francine B. Perler |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Mycobacterium xenopi
Recombinant Fusion Proteins Saccharomyces cerevisiae Tropomyosin Protein Engineering Cleavage (embryo) DNA gyrase General Biochemistry Genetics and Molecular Biology Thioredoxins Bacterial Proteins Protein splicing Escherichia coli Protein Splicing Cloning Molecular biology Temperature Protein engineering biology.organism_classification Fusion protein DNA Topoisomerases Type II Mannose-Binding Lectins Biochemistry DNA Gyrase Carrier Proteins Intein Biotechnology Cysteine |
| Zdroj: | BioTechniques. 27:110-120 |
| 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: | OpenAIRE |
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