Papillomavirus capsid protein expression in Escherichia coli : purification and assembly of HPV11 and HPV16 L1 1 1 Edited by M. Yaniv

Papillomavirus capsid protein expression in Escherichia coli : purification and assembly of HPV11... 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 (ΔN10) and 30 residues from the carboxy terminus (ΔC30) 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 ΔN10, 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

Papillomavirus capsid protein expression in Escherichia coli : purification and assembly of HPV11 and HPV16 L1 1 1 Edited by M. Yaniv

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Publisher
Elsevier
Copyright
Copyright © 2001 Academic Press
ISSN
0022-2836
DOI
10.1006/jmbi.2000.4464
Publisher site
See Article on Publisher Site

Abstract

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 (ΔN10) and 30 residues from the carboxy terminus (ΔC30) 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 ΔN10, 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.

Journal

Journal of Molecular BiologyElsevier

Published: Mar 16, 2001

References

  • Conserved features in papillomavirus and polyomavirus capsids
    Belnap, D.M.; Olson, N.H.; Cladel, N.M.; Newcomb, W.W.; Brown, J.C.; Kreider, J.W.; Christensen, N.D.; Baker, T.S.
  • Prevalence of human papillomavirus in cervical cancer
    Bosch, F.X.; Manos, M.M.; Munoz, N.; Sherman, M.; Jansen, A.M.; Peto, J.; Schiffman, M.H.; Moreno, V.; Kurman, R.; Shah, K.V.
  • Estimates of the worldwide mortality from eighteen major cancers in 1985. Implications for prevention and projections of future burden
    Pisani, P.; Parkin, D.M.; Ferlay, J.

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