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Applied and Environmental Microbiology, May 2008, p. 3099-3104, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.00305-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Shuttle Vector Expression in Thermococcus kodakaraensis: Contributions of cis Elements to Protein Synthesis in a Hyperthermophilic Archaeon

Thomas J. Santangelo, L'ubomíra uboová, and John N. Reeve^*

Department of Microbiology, Ohio State University, Columbus, Ohio 43210

Received 5 February 2008/ Accepted 19 March 2008

Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T. kodakaraensis transformants could be selected by trpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle vector expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit of T. kodakaraensis RNA polymerase (RNAP), was 8-fold higher than chromosome expression. An idealized ribosome binding sequence (5'-AGGTGG) was incorporated for RpoL-HA expression, and changes to this sequence reduced expression. Changing the translation initiation codon from AUG to GUG did not reduce RpoL-HA expression, but replacing AUG with UUG dramatically reduced RpoL-HA synthesis. When functioning as translation initiation codons, AUG, GUG, and UUG all directed the incorporation of methionine as the N-terminal residue of RpoL-HA synthesized in T. kodakaraensis. Affinity purification confirmed that an HA- plus six-histidine-tagged RpoL subunit (RpoL-HA-his₆) synthesized ectopically from a shuttle vector was assembled in vivo into RNAP holoenzymes that were active and could be purified directly from T. kodakaraensis cell lysates by Ni²⁺ binding and imidazole elution.

Published ahead of print on 31 March 2008.

Permanent address: Institute of Animal Biochemistry and Genetics, Ivanka Pri Dunayi, Slovak Republic.