Expression of the human papillomavirus type 11 L1 capsid protein in Escherichia coli: characterization of protein domains involved in DNA binding and capsid assembly.
The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to assemble further. In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells. The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein. The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.
Li
M
M
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262, USA.
Cripe
T P
TP
Estes
P A
PA
Lyon
M K
MK
Rose
R C
RC
Garcea
R L
RL
D000595:Amino Acid Sequence; D001665:Binding Sites; D002213:Capsid / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D027383:Papillomaviridae / Q000235:genetics* / Q000502:physiology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D019065:Virus Assembly
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; C094776:L1 protein, Human papillomavirus type 11; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D014357:Trypsin