Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression

Protein Expr Purif. 2017 May:133:193-198. doi: 10.1016/j.pep.2016.06.010. Epub 2016 Jun 25.

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.

Keywords: Actin-binding protein; Escherichia coli; Recombinant protein expression; Sarkosyl detergent; Truncated mutant.

MeSH terms

  • Dictyostelium / genetics*
  • Dictyostelium / metabolism
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Microfilament Proteins* / biosynthesis
  • Microfilament Proteins* / chemistry
  • Microfilament Proteins* / genetics
  • Protozoan Proteins* / biosynthesis
  • Protozoan Proteins* / chemistry
  • Protozoan Proteins* / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics

Substances

  • Microfilament Proteins
  • Protozoan Proteins
  • Recombinant Proteins