Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells.

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Zhang Z, Alfonta L, Tian F, Bursulaya B, Uryu S, King DS, Schultz PG

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells.

Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):8882-7. doi: 10.1073/pnas.0307029101. Epub 2004 Jun 8.

PubMed ID

15187228 [ View in PubMed

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Abstract

An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA(Trp) (mutRNA(UCA)(Trp)) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNA(Trp) was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNA(UCA)(Trp) gene was inserted between the 5' and 3' flanking sequences of the tRNA(Trp-1) gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate mutRNA(UCA)(Trp) and no endogenous mammalian tRNAs. Similarly, the mutRNA(UCA)(Trp) is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-l-tryptophan. The resulting mutant BsTrpRS-mutRNA(UCA)(Trp) pair allows the efficient and selective incorporation of 5-hydroxy-l-tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Oxitriptan	Tryptophan--tRNA ligase 2	Protein	Deinococcus radiodurans (strain ATCC 13939 / DSM 20539 / JCM 16871 / LMG 4051 / NBRC 15346 / NCIMB 9279 / R1 / VKM B-1422)	Unknown	Not Available	Details