Isolation and Crystallization of a Chimeric Qβ Replicase Containing Thermus thermophilus EF-Ts

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N. N. Vasiliev¹, L. Jenner^2,3, M. M. Yusupov², and A. B. Chetverin^1*

¹Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; E-mail: alexch@vega.protres.ru

²Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France; E-mail: marat@igbmc.fr

³INSERM, U964, Illkirch, F-67400, France

^* To whom correspondence should be addressed.

Received March 13, 2010; Revision received April 8, 2010

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Qβ replicase is a protein complex responsible for the replication of the genomic RNA of bacteriophage Qβ. In addition to the phage-encoded catalytic β subunit, it recruits three proteins from the host Escherichia coli cell: elongation factors EF-Tu and EF-Ts and ribosomal protein S1. We prepared a chimeric Qβ replicase in which the E. coli EF-Ts is replaced with EF-Ts from Thermus thermophilus. The chimeric protein is produced in E. coli cells during coexpression of the genes encoding the β subunit and thermophilic EF-Ts. The developed isolation procedure yields a substantially homogeneous preparation of the chimeric replicase. Unlike the wild-type enzyme, the S1-less chimeric replicase could be crystallized. This result facilitates studies on the structure of Qβ replicase and the mechanism of recognition of its templates that can replicate in vitro at a record rate.

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KEY WORDS: RNA replication, RNA-dependent RNA polymerase, ribosomal protein S1, elongation factors EF-Tu and EF-Ts, two plasmid expression system, protein crystallization, structure stability

DOI: 10.1134/S0006297910080067

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