Design, expression, and purification of a Flaviviridae polymerase using a high-throughput approach to facilitate crystal structure determination

Kyung Choi, James M. Groarke, Dorothy C. Young, Michael G. Rossmann, Daniel C. Pevear, Richard J. Kuhn, Janet L. Smith

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical "core," although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized.

Original languageEnglish (US)
Pages (from-to)2685-2692
Number of pages8
JournalProtein Science
Volume13
Issue number10
DOIs
StatePublished - Oct 2004
Externally publishedYes

Fingerprint

Flaviviridae
Bovine Viral Diarrhea Viruses
Viruses
Purification
Crystal structure
Throughput
Proteolysis
Proteins
RNA Replicase
Mutagenesis
Crystallization
Hepacivirus
Trypsin
Escherichia coli
Clone Cells
Sampling
Crystals

Keywords

  • Bovine viral diarrhea virus
  • Flaviviridae
  • Hepatitis C virus
  • High-throughput
  • Limited proteolysis
  • NS5B
  • RNA-dependent RNA polymerase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Design, expression, and purification of a Flaviviridae polymerase using a high-throughput approach to facilitate crystal structure determination. / Choi, Kyung; Groarke, James M.; Young, Dorothy C.; Rossmann, Michael G.; Pevear, Daniel C.; Kuhn, Richard J.; Smith, Janet L.

In: Protein Science, Vol. 13, No. 10, 10.2004, p. 2685-2692.

Research output: Contribution to journalArticle

Choi, Kyung ; Groarke, James M. ; Young, Dorothy C. ; Rossmann, Michael G. ; Pevear, Daniel C. ; Kuhn, Richard J. ; Smith, Janet L. / Design, expression, and purification of a Flaviviridae polymerase using a high-throughput approach to facilitate crystal structure determination. In: Protein Science. 2004 ; Vol. 13, No. 10. pp. 2685-2692.
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