Papillomavirus capsid protein expression in Escherichia coli: purification and assembly of HPV11 and HPV16 L1.
The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.
Department of Biochemistry & Molecular Genetics, University of Colorado School of Medicine, 4200 E. 9th Ave, Denver, CO 80262, USA. xiaojiang.chen@uchsc.edu
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Chen
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Casini
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Harrison
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Garcea
D001704:Biopolymers / Q000737:chemistry; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D027383:Papillomaviridae / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
D001704:Biopolymers; D036022:Capsid Proteins; C094776:L1 protein, Human papillomavirus type 11; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16