Production of human β-actin and a mutant using a bacterial expression system with a cold shock vector.
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 β-actin was successfully expressed using a cold shock vector, pCold, in the bacterial expression system. The expressed β-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 β-actin showed a normal polymerization ability compared with β-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.
Tamura
Minoru
M
Department of Applied Chemistry, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan. Tamura@eng.ehime-u.ac.jp
Ito
Katsunori
K
Kunihiro
Sachio
S
Yamasaki
Chihoko
C
Haragauchi
Mihoko
M
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D015153:Blotting, Western; D002846:Chromatography, Affinity; D057773:Cold Shock Proteins and Peptides / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D000199:Actins; D057773:Cold Shock Proteins and Peptides; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine