Stereoselective Incorporation of an Unsaturated Isoleucine Analogue into a Protein Expressed in E. coli

✍ Scribed by Marissa L. Mock; Thierry Michon; Jan C. M. van Hest; David A. Tirrell

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 131 KB
• Volume: 7
• Category: Article
• ISSN: 1439-4227
• DOI: 10.1002/cbic.200500201

✦ Synopsis

Abstract

The unsaturated amino acid 2‐amino‐3‐methyl‐4‐pentenoic acid (E‐Ile) was prepared in the form of its (2__S,3__S__),(2__R__,3__R__) and (2__S__,3__R__),(2__R__,3__S__) stereoisomeric pairs. The translational activities of__ SS__‐E‐Ile and__ SR__‐E‐Ile were assessed in an__ E. coli strain rendered auxotrophic for isoleucine. SS__‐E‐Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %;__ SR__‐E‐Ile yielded no conclusive evidence for incorporation. ATP/PP~i~__ exchange assays indicated that SS__‐E‐Ile was activated by the isoleucyl‐tRNA synthetase at a rate comparable to that characteristic of isoleucine;__ SR__‐E‐Ile was activated approximately 100‐times more slowly than__ SS__‐E‐Ile.__