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

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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Tomar S,Narwal M,Harms E,Smith JL,Kuhn RJ

doi

10.1016/j.pep.2011.05.022

subject

Has Abstract

pub_date

2011-10-01 00:00:00

pages

277-84

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(11)00147-1

journal_volume

79

pub_type

杂志文章

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