Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli
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The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.
KeywordsInfectious hematopoietic necrosis virus Heterologous protein expression Nucleoprotein Osmolytes Purification Protein solubility
The authors acknowledge the financial support of this study by Iran Vice-Presidency for Science and Technology and Institute of Biotechnology and Bioengineering of Isfahan University of Technology.
Compliance with ethical standards
Conflict of interest
The authors have declared no conflict of interest.
All applicable international, national, and institutional guidelines for the care and use of animals were followed. This manuscript does not contain any studies with human participants performed by any of the authors.
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