- Efficient incorporation of unsaturated methionine analogues into proteins in vivo
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A set of eight methionine analogues was assayed for translational activity in Escherichia coli. Norvaline and norleucine, which are commercially available, were assayed along with 2-amino-5-hexenoic acid (2), 2-amino-5-hexynoic acid (3), cis-2-amino-4-hexenoic acid (4), trans-2-amino-4-hexenoic acid (5), 6,6,6-trifluoro-2-aminohexanoic acid (6), and 2-aminoheptanoic acid (7), each of which was prepared by alkylation of diethyl acetamidomalonate with the appropriate tosylate, followed by hydrolysis. The E. coli methionine auxotroph CAG18491, transformed with plasmids pREP4 and pQE15, was used as the expression host, and translational activity was assayed by determination of the capacity of the analogue to support synthesis of the test protein dihydrofolate reductase (DHFR) in the absence of added methionine. The importance of amino acid side chain length was illustrated by the fact that neither norvaline (8) nor 7 showed translational activity, in contrast to norleucine (9), which does support protein synthesis under the assay conditions. The internal alkene functions of 4 and 5 prevented incorporation of these analogues into test protein, and the fluorinated analogue 6 yielded no evidence of translational activity. The terminally unsaturated compounds 2 and 3, however, proved to be excellent methionine surrogates: 1H NMR spectroscopy, amino acid analysis, and N-terminal sequencing indicated ~85% substitution of methionine by 2, while 3 showed 90-100% replacement. Both analogues also function efficiently in the initiation step of protein synthesis, as shown by their near-quantitative occupancy of the N-terminal amino acid site in DHFR. Enzyme kinetics assays were conducted to determine the rate of activation of each of the methionine analogues by methionyl tRNA synthetase (MetRS); results of the in vitro assays corroborate the in vivo incorporation results, suggesting that success or failure of analogue incorporation in vivo is controlled by MetRS.
- Van Hest, Jan C. M.,Kiick, Kristi L.,Tirrell, David A.
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p. 1282 - 1288
(2007/10/03)
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- Protein engineering by in vivo incorporation of non-natural amino acids: Control of incorporation of methionine analogues by methionyl-tRNA synthetase
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The incorporation of non-natural amino acids is an important strategy for engineering novel chemical and physical properties into natural and artificial proteins. The incorporation of amino acids into proteins in vivo is controlled in large part by the aminoacyl-tRNA synthetases (AARS). We have measured kinetic constants for in vitro activation of a set of methionine analogues by methionyl-tRNA synthetase (MetRS) via the ATP-PP(i) exchange reaction. Activation of methionine analogues in vitro correlates well with the ability of these analogues to support protein synthesis in vivo, substantiating the critical role of the AARS in controlling the incorporation of non-natural amino acids into proteins. Methionine analogues with k(cat)/K(m) values 2000-fold lower than those for methionine can support synthesis of a typical target protein (mDHFR) under standard conditions of protein expression. The kinetic constants correlate well with observed protein yields from a conventional bacterial expression host, indicating that the MetRS activity of the host can control the level of protein synthesis under certain conditions. Furthermore, increasing the MetRS activity of the bacterial host results in increased protein synthesis in media supplemented with the methionine analogues homoallylglycine and norleucine. These results suggest new strategies for incorporation of non-natural amino acids via manipulation of the AARS activity of a bacterial host. (C) 2000 Elsevier Science Ltd.
- Kiick, Kristi L.,Tirrell, David A.
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p. 9487 - 9493
(2007/10/03)
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