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

• Published in: Protein expression and purification Vol. 48; no. 1; pp. 14 - 23
• Main Authors: Ivanov, Alexander V., Korovina, Anna N., Tunitskaya, Vera L., Kostyuk, Dmitry A., Rechinsky, Vladimir O., Kukhanova, Marina K., Kochetkov, Sergey N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-07-2006
• Subjects: Amino Acid Sequence; Base Sequence; Casein Kinase II - metabolism; Codon, Initiator; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Genes; Genetic Vectors; Hepacivirus - genetics; Hepacivirus - metabolism; Hepatitis C virus; High-level expression; Histidine - genetics; Histidine - metabolism; Humans; Models, Genetic; Molecular Sequence Data; Plasmids - genetics; Plasmids - metabolism; Point Mutation; Protein Engineering - methods; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA-dependent RNA polymerase; Viral Nonstructural Proteins - biosynthesis; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - isolation & purification; Hepatitis C virus; High-level expression; RNA-dependent RNA polymerase
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.02.011

The plasmid pET-21d-2c-5BΔ55 effectively expressing a C-terminally truncated form (NS5BΔ55) of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed. It was derived from pET-21d-5BΔ55 plasmid and contained six mutations in the ATG-start codon region and an additional cistron upstream the target gene. The C-terminally His-tagged NS5BΔ55 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 NS5BΔ55 protein expressed from the parental pET-21d-5BΔ55 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-5BΔ55 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 NS5BΔ55 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.