Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 78, Issue 1, Pages 1-5Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2010.09.007
Keywords
beta-Actin; pCold; Bacterial expression; Polymerization; Mutation
Categories
Funding
- [19510219]
- [21510227]
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Actin is the most abundant protein in the cytoplasm of most eukaryotic cells and is involved in a variety of cellular functions. It has been difficult to produce actin in bacterial expression systems in good yields. In this study, we developed a new simple method for the production of recombinant actin in Escherichia coli cells. Human beta-actin was successfully expressed using a cold shock vector, pCold, in the bacterial expression system. The expressed beta-actin (hexahistidine-tagged) was separated with a Ni-chelating resin, followed by a polymerization/depolymerization cycle or column chromatography with the Ni-chelating resin. The purified recombinant beta-actin showed a normal polymerization ability compared with beta-actin purified from human platelets. We produced a recombinant mutant actin with a Gly-168Arg mutation in the system and confirmed that it exhibited an impaired polymerization ability. The system developed in this study will provide a useful method for the production of actin isoforms and their mutants. (C) 2011 Published by Elsevier Inc.
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