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Title: Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1
Authors: Tomar, Shailly
Narwal M.
Harms E.
Smith J.L.
Kuhn R.J.
Published in: Protein Expression and Purification
Abstract: Alphavirus nonstructural protein nsP1 possesses distinct methyltransferase (MTase) and guanylyltransferase (GTase) activities involved in the capping of viral RNAs. In alphaviruses, the methylation of GTP occurs before RNA transguanylation and nsP1 forms a covalent complex with m 7GMP unlike the host mRNA guanylyltransferase which forms GMP-enzyme complex. In this study, full length SINV nsP1 was expressed in a soluble form with an N-terminal histidine tag in Escherichia coli and purified to homogeneity. The purified protein is enzymatically active and contains both MTase and GTase activity indicating that SINV nsP1 does not require membrane association for its enzymatic function. Biochemical analysis shows that detergents abolish nsP1 GTase activity, whereas nonionic detergents do not affect MTase activity. Furthermore, SINV nsP1 contains the metal-ion dependent GTase, whereas MTase does not require a metal ion. Circular dichroism spectroscopic analysis of purified protein indicate that nsP1 has a mixed ?/? structure and is in the folded native conformation. © 2011 Elsevier Inc. All rights reserved.
Citation: Protein Expression and Purification(2011), 79(2): 277-284
Issue Date: 2011
Keywords: Alphavirus
ISSN: 10465928
Author Scopus IDs: 57203506001
Author Affiliations: Tomar, S., Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, India
Narwal, M., Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, India
Harms, E., Department of Biological Sciences, Bindley Bioscien
Funding Details: The authors thank Steven S. Broyles, Purdue University, for the kind gift of vaccinia virus capping enzyme, and James Strauss, California Institute of Technology, for the kind gift of anti-SINV nsP1 antibody, and Anita Robinson for assistance with the man
Corresponding Author: Kuhn, R.J.; Department of Biological Sciences, Hockmeyer Hall of Structural Biology, Purdue University, 240 S. Martin Jischke Dr., West Lafayette, IN 47907, United States; email:
Appears in Collections:Journal Publications [BT]

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