Expression and purification of rotavirus proteins NSP5 and NSP6 in Escherichia coli.

Abstract:

:Rotaviruses are one of the worldwide leading causes of gastroenteritis in children under 5 yr old. The rotavirus nonstructural NSP5 is a phosphoprotein implicated in viroplasms formation, whereas NSP6 could have a possible regulatory role of NSP5. It has been reported that N- and C-termini of NSP5 are important for their function. However, no structural information on NSP5 and NSP6 proteins is available. Because a high amount of protein is required for structural analysis, efficient expression systems are required. His-tag fusion at the C-terminus and glutathione-S-transferase (GST)-fusion at the N-terminus were used as expression systems, and conditions for recombinant proteins expression were obtained. His-tag fusion was not efficient to produce NSP5 (2% of total protein), but NSP6 was expressed in higher amounts (11% of total protein). In contrast, GSTNSP5 and GST-NSP6 proteins correspond to 34 and 31% of the total proteins, respectively. GST-fusions seem to have a protective effect against nonstructural rotavirus protein toxicity in Escherichia coli; however, in both systems, NSP5 and NSP6 recombinant proteins were expressed as inclusion bodies. Conditions for solubilization and purification of recombinant proteins were achieved. This is the first report of expression and purification of NSP5 and NSP6 recombinant proteins in suitable amounts for further structural analysis.

journal_name

Cell Biochem Biophys

authors

Samaniego-Hernández M,De León-Rodriguez A,Aparicio-Fabre R,Arias-Ortiz C,Barba de la Rosa AP

doi

10.1385/CBB:44:3:336

subject

Has Abstract

pub_date

2006-01-01 00:00:00

pages

336-41

issue

3

eissn

1085-9195

issn

1559-0283

pii

CBB:44:3:336

journal_volume

44

pub_type

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