Design, expression, and purification of a Flaviviridae polymerase using a high-throughput approach to facilitate crystal structure determination.
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.
Choi
Kyung H
KH
Department of Biological Sciences, Purdue University, 915 West State St., West Lafayette, IN 47907-2054, USA.
Groarke
James M
JM
Young
Dorothy C
DC
Rossmann
Michael G
MG
Pevear
Daniel C
DC
Kuhn
Richard J
RJ
Smith
Janet L
JL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003460:Crystallization; D003461:Crystallography; D001908:Diarrhea Viruses, Bovine Viral / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014357:Trypsin / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase; D014357:Trypsin