title: Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins.
abstract: 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.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991, 32 Vavilov str., Moscow, Russian Federation. aivanov@yandex.ru
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Ivanov
authors.forename: Anna N
authors.initials: AN
authors.lastname: Korovina
authors.forename: Vera L
authors.initials: VL
authors.lastname: Tunitskaya
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Kostyuk
authors.forename: Vladimir O
authors.initials: VO
authors.lastname: Rechinsky
authors.forename: Marina K
authors.initials: MK
authors.lastname: Kukhanova
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Kochetkov
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D047390:Casein Kinase II / Q000378:metabolism; D018387:Codon, Initiator; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D015202:Protein Engineering / Q000379:methods; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012324:RNA-Dependent RNA Polymerase
chemical_list: D047390:Casein Kinase II; D018387:Codon, Initiator; D006639:Histidine; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase