title: Expression of the HSV-1 capsid protein VP19C in Escherichia coli: a single amino acid change overcomes an expression block of the full-length polypeptide.
abstract: The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.
authors.affiliation: Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University, Rm. 353, CRB1, 1650 Orleans Street, Baltimore, MD 21231, USA.
authors.forename: Brandon W
authors.initials: BW
authors.lastname: Henson
authors.forename: Nicole
authors.initials: N
authors.lastname: Johnson
authors.forename: Alakesh
authors.initials: A
authors.lastname: Bera
authors.forename: Mercy E
authors.initials: ME
authors.lastname: Okoye
authors.forename: Keshal Viren
authors.initials: KV
authors.lastname: Desai
authors.forename: Prashant J
authors.initials: PJ
authors.lastname: Desai
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D018258:Herpesvirus 2, Human / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; C063128:ICP32-VP19c capsid protein, herpes simplex virus type 2; D011994:Recombinant Proteins