Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies

J Biotechnol. 2013 Dec;168(4):506-10. doi: 10.1016/j.jbiotec.2013.09.019. Epub 2013 Oct 4.

Abstract

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies.

Keywords: GST; GST-tagged proteins; Inclusion bodies; Urea.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens / biosynthesis*
  • Allergens / genetics
  • Animals
  • Colorimetry
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial
  • Glutathione Transferase / genetics*
  • Glutathione Transferase / metabolism
  • Inclusion Bodies / enzymology*
  • Mice
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics*
  • Urea / chemistry
  • Urea / metabolism

Substances

  • Allergens
  • Recombinant Fusion Proteins
  • Urea
  • Glutathione Transferase