Expression, purification, and functional characterization of a stable helicase domain from a tomato mosaic virus replication protein

• Published in: Protein expression and purification Vol. 81; no. 1; pp. 89 - 95
• Main Authors: Xiang, Hongyu, Ishibashi, Kazuhiro, Nishikiori, Masaki, Jaudal, Mauren C., Ishikawa, Masayuki, Katoh, Etsuko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-01-2012
• Subjects: 5′-Triphosphatase tobacco mosaic virus; Adenosine Triphosphate - metabolism; Alphavirus-like superfamily; Amino Acid Sequence; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - genetics; Helicase domain; Molecular Sequence Data; Protein Stability; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Replication protein; RNA Helicases - chemistry; RNA Helicases - genetics; RNA Helicases - metabolism; Tobamovirus; Tobamovirus - enzymology; Tobamovirus - genetics; Tomato mosaic virus; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Tobamovirus; Alphavirus-like superfamily; Replication protein; Helicase domain; Tomato mosaic virus; 5′-Triphosphatase tobacco mosaic virus
• DOI: 10.1016/j.pep.2011.09.002
• EISSN: 1096-0279
• ISSN: 1046-5928

► We expressed the ToMV replication proteins that contain the helicase domain. ► We determined the stable helicase fragment. ► Its N-terminal part was necessary for NTPase activity and stability. ► We developed an efficient expression and purification procedures for the fragment. ► The stable fragment has nucleoside 5′-triphosphatase activity.
Tomato mosaic virus (genus, Tobamovirus) is a member of the alphavirus-like superfamily of positive-strand RNA viruses, which include many plant and animal viruses of agronomical and clinical importance. The RNA of alphavirus-like superfamily members encodes replication-associated proteins that contain a putative superfamily 1 helicase domain. To date, a viral three-dimensional superfamily 1 helicase structure has not been solved. For the study reported herein, we expressed tomato mosaic virus replication proteins that contain the putative helicase domain and additional upstream N-terminal residues in Escherichia coli. We found that an additional 155 residues upstream of the N-terminus of the helicase domain were necessary for stability. We developed an efficient procedure for the expression and purification of this fragment and have examined factors that affect its stability. Finally, we also showed that the stable fragment has nucleoside 5′-triphosphatase activity.