Format

Send to

Choose Destination
Protein Expr Purif. 2011 Oct;79(2):277-84. doi: 10.1016/j.pep.2011.05.022. Epub 2011 Jun 13.

Heterologous production, purification and characterization of enzymatically active Sindbis virus nonstructural protein nsP1.

Author information

1
Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, India. shailfbt@iitr.ernet.in

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(7)GMP 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 α/β structure and is in the folded native conformation.

PMID:
21693190
PMCID:
PMC3155615
DOI:
10.1016/j.pep.2011.05.022
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center