Purification and structural characterization of herpes simplex virus glycoprotein C
Purification of herpes simplex virus glycoprotein C (gC) in microgram amounts yielded sufficient material for an analysis of its secondary structure. Purification was facilitated by using the mutant virus gC-3, which bears a point mutation that interrupts the putative hydrophobic membrane anchor sequence, causing the secretion of gC-3 protein into the cell culture medium. gC-3 protein was purified by size fractionation of concentrated culture medium from infected cells on a gel filtration column of Sephacryl S-200, followed by immunoaffinity chromatography on a column constructed of gC-specific monoclonal antibodies cross-linked to a protein A-Sepharose CL-4B matrix. Purified gC-3 had a molecular weight of 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size expected for gC, was reactive with gC-specific monoclonal antibodies in protein immunoblots, and contained amino acid sequences characteristic of gC as determined by radiochemical amino acid microsequence analyses. Polyclonal antisera obtained from a rabbit immunized with gC-3 reacted with wild-type gC in immunoprecipitation, enzyme immunoassay, and immunoelectroblot (western blot) assays. Deglycosylation by treatment with trifluoromethanesulfonic acid reduced the molecular weight of gC-3 by approximately 35%. Analyses of both native and deglycosylated gC-3 by Raman spectroscopy showed that the native molecule consists of about 17%..cap alpha..-helix, 24% ..beta..-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% ..cap alpha..-helix, 10% ..beta..-sheet, 81% disordered structures. These data were in good agreement with the 11% ..cap alpha..-helix, 18% ..beta..-sheet, 61% ..beta..-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3.
- Research Organization:
- Univ. of Michigan, Ann Arbor
- OSTI ID:
- 6790630
- Journal Information:
- Biochemistry; (United States), Vol. 26:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
GLUCOPROTEINS
MOLECULAR STRUCTURE
PURIFICATION
RADIOASSAY
AMINO ACID SEQUENCE
ELECTROPHORESIS
ENZYME IMMUNOASSAY
HERPES SIMPLEX
IMMUNE SERUMS
ION EXCHANGE CHROMATOGRAPHY
MOLECULAR WEIGHT
PRECIPITATION
VIRUSES
CARBOHYDRATES
CHROMATOGRAPHY
DISEASES
IMMUNOASSAY
INFECTIOUS DISEASES
MICROORGANISMS
ORGANIC COMPOUNDS
PARASITES
PROTEINS
SACCHARIDES
SEPARATION PROCESSES
SKIN DISEASES
VIRAL DISEASES
550601* - Medicine- Unsealed Radionuclides in Diagnostics