title: Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.
abstract: Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression.
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, 1, Kangwondaehak-gil, Chuncheon-si, Gangwon-do, 24341, South Korea.
authors.forename: Jeong Min
authors.initials: JM
authors.lastname: Chung
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, 1, Kangwondaehak-gil, Chuncheon-si, Gangwon-do, 24341, South Korea.
authors.forename: Sangmin
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, 1, Kangwondaehak-gil, Chuncheon-si, Gangwon-do, 24341, South Korea. Electronic address: hsjung@kangwon.ac.kr.
authors.forename: Hyun Suk
authors.initials: HS
authors.lastname: Jung
mesh_terms: D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D008840:Microfilament Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins
keywords: Actin-binding protein; Escherichia coli; Recombinant protein expression; Sarkosyl detergent; Truncated mutant