Improved purification protocol of the HSV-1 protease catalytic domain, using immunoaffinity.
The catalytic domain of herpes simplex virus protease was expressed in baculovirus-infected cells and purified in milligram quantities by ion-exchange and size-exclusion chromatography. The usefulness of this material was limited by the presence of a contaminating proteolytic activity, which caused time-dependent degradation of the protease. As a result we decided to explore an alternative approach to purification. Specific monoclonal antibodies were produced and evaluated by surface plasmon resonance as ligands for immunoaffinity chromatography. One monoclonal antibody, 6H4, was chosen for coupling to an affinity support, and the resulting column allowed us to obtain a pure and stable enzyme. Immunoaffinity chromatography of herpes simplex virus type 1 protease resulted in successful elimination of the contaminating protease activity. Moreover the immunoaffinity column permitted the isolation of stable and pure enzyme in a one-column procedure.
McKercher
G
G
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Laval, QC, Canada.
Bonneau
P R
PR
Lagacé
L
L
Thibeault
D
D
Massariol
M J
MJ
Krogsrud
R
R
Lawetz
C
C
McDonald
P C
PC
Cordingley
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D002213:Capsid / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D007163:Immunosorbent Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D014764:Viral Proteins
D000911:Antibodies, Monoclonal; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D012697:Serine Endopeptidases