Use of the Methyl Ester of a Fluorescent Unnatural Amino Acid to Facilitate Site-Specific Incorporation of Fluorescent Probes in Proteins

Abstract

Fluorescent unnatural amino acids (UAAs) can be efficiently incorporated in target proteins in vivo by expressing suppressor tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pairs in the presence of target protein mRNA that contains an orthogonal stop codon in the coding sequence and adequate cellular levels of UAA. While conceptually straightforward, many technical barriers impede facile application of this technology in a broad array of eukaryotic expression systems. Cell loading of UAAs is one such obstacle since amino acids usually require specific transporters for cellular uptake. To improve UAA cell loading, the methyl ester of one fluorescent UAA, L-Anap (L-Anap-AM) was tested in eukaryotic expression systems. L-Anap-AM is soluble in ethanol and DMSO, and readily diluted into aqueous solutions. Full length GFP (containing a stop codon mutation at Y39) and AHA2 H+-ATPase (stop codon mutation at W71) were produced in yeast strains expressing a tRNA/aaRS pair for L-Anap after growth in L-Anap-AM containing media. Both expressed proteins were fluorescent and GFP showed efficient FRET between L-Anap and the protein fluorochrome. LC/MS/MS studies also showed that L-Anap was located at residue 39 in GFP. These studies demonstrated that L-Anap-AM is correctly incorporated into peptide chains during translation. Studies were also carried out in Xenopus oocytes in which nuclear injection of the tRNA/aaRS pair for L-Anap was followed by injection of cRNA for Connexin 26 (Cx26) or the Shaker KV channel containing a stop codon mutation at specific locations. Both Cx26 and K+ currents were measured in injected oocytes, using a two-microelectrode voltage clamp, only after incubation in an L-Anap-AM containing storage buffer. These studies demonstrate that L-Anap-AM can be used effectively to generate UAA-containing proteins in a variety of eukaryotic expression systems.