Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins.

The plasmid pET-21d-2c-5BDelta55 effectively expressing a C-terminally truncated form (NS5BDelta55) of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed. It was derived from pET-21d-5BDelta55 plasmid and contained six mutations in the ATG-start codon region and an additional cistron upstream the target gene. The C-terminally His-tagged NS5BDelta55 protein was expressed in Rosetta(DE3) Escherichia coli strain bearing an additional pRARE plasmid encoding extra copies of rare tRNAs. The yield of the target enzyme exceeded by a factor of 29 the yield of NS5BDelta55 protein expressed from the parental pET-21d-5BDelta55 plasmid (5 mg/L). The increase in the protein yield could be explained by facilitated protein translation initiation, resulted from disruption of the stable secondary mRNA structure. The pET-21d-2c-5BDelta55 plasmid yielded one third amount of the protein when expressed in BL-21(DE3) strain, indicating that the pRARE plasmid is required for a high-level expression of NS5BDelta55 protein. The 29-fold enhancement of the protein yield was accompanied by only a 2.5-fold increase of the corresponding mRNA level. The expression of another HCV NS5A protein His-tagged at the C-terminus in the developed system yielded a similar amount of the protein (4 mg/L), whereas its N-terminally His-tagged counterpart was obtained in a 30 mg/L yield. The NS5A protein purified under denaturing conditions and renatured in solution inhibited the HCV RdRp and was a substrate for human casein kinase II.