GB virus C E2 glycoprotein: expression in CHO cells, purification and characterization.
A 315 amino acid recombinant segment of the GB virus C (GBV-C) E2 envelope glycoprotein (E2-315) was expressed and secreted from CHO cells. E2-315 was purified by affinity chromatography using a monoclonal antibody directed to a FLAG sequence genetically engineered onto the C terminus of the recombinant protein. The secreted protein had a molecular mass of 48-56 kDa and was shown to be N-glycosylated. Amino acid sequencing confirmed the expected N-terminal sequence. Purified E2-315 was used to develop an ELISA for detection of E2 antibodies in human sera. Antibodies to GBV-C E2 appeared to be directed toward conformational epitopes since human sera reactivity was detected in ELISA using native E2-315, but it was extremely weak or non-existent with denatured E2 protein. The use of an ELISA which can detect human GBV-C E2 antibodies will be important in further understanding of the clinical significance and epidemiology of GBV-C.
Surowy
T K
TK
Experimental Biology Research, Abbott Laboratories, Dept. 90D, North Chicago, IL 60064, USA. teresa.surowy@add.ssw.abbott.com
Leary
T P
TP
Carrick
R J
RJ
Knigge
M F
MF
Pilot-Matias
T J
TJ
Heynen
C
C
Gutierrez
R A
RA
Desai
S M
SM
Dawson
G J
GJ
Mushahwar
I K
IK
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D018067:Flaviviridae / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006508:Hepatitis Antibodies / Q000097:blood*; D006525:Hepatitis, Viral, Human / Q000175:diagnosis / Q000453:epidemiology / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D006508:Hepatitis Antibodies; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C107619:glycoprotein E2, GB virus C