Selective Incorporation of 5-Hydroxytryptophan into Proteins in Mammalian Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 2004-06, Vol.101 (24), p.8882-8887
• Main Authors: Zhang, Zhiwen, Alfonta, Lital, Tian, Feng, Bursulaya, Badry, Uryu, Sean, King, David S., Schultz, Peter G., Wells, James A.
• Format: Article
• Language: English
• Subjects: 5-Hydroxytryptophan - metabolism; Amino acids; Antibodies; Anticodon - genetics; Arabidopsis - genetics; Arabidopsis thaliana; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Base Sequence; Biochemistry; Biological Sciences; Cell Line; Codon - genetics; Codons; Cross-Linking Reagents - chemistry; Electrodes; Escherichia coli - metabolism; Fluorescence; Genes; Genetic mutation; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nucleic Acid Conformation; Opal; Plasmids; Proteins; RNA, Transfer, Trp - genetics; RNA, Transfer, Trp - metabolism; Spectrometry, Fluorescence; Suppression, Genetic; Transfection; Transfer RNA; Tryptophan-tRNA Ligase - chemistry; Tryptophan-tRNA Ligase - genetics; Tryptophan-tRNA Ligase - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0307029101

An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNATrp( mutRNAUCA Trp) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtills tRNATrpwas mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNAUCA Trpgene was inserted between the 5′ and 3′ flanking sequences of the tRNATrp-1gene 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 mutRNAUCA Trpand no endogenous mammalian tRNAs. Similarly, the mutRNAUCA Trpis 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- mutRNAUCA Trppair 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.