Site-specific incorporation of tryptophan analogues into recombinant proteins in bacterial cells.
A designed yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) activates four tryptophan (Trp) analogues (6-chlorotryptophan (6ClW), 6-bromotryptophan (6BrW), 5-bromotryptophan (5BrW), and benzothienylalanine (BT)) that are not utilized by the endogenous E. coli translational apparatus. Introduction of yPheRS (T415G) and a mutant yeast phenylalanine amber suppressor tRNA (ytRNAPheCUA_UG) into an E. coli expression host allowed site-specific incorporation of three of these analogues (6ClW, 6BrW, and BT) into recombinant murine dihydrofolate reductase in response to amber stop codons with at least 98% fidelity. All three analogues were introduced at the Trp66 position in the chromophore of a cyan fluorescent protein variant (CFP6) to investigate the attendant changes in spectral properties. Each of the analogues caused blue shifts in the fluorescence emission and absorption maxima. The CFP6 variant bearing BT at position 66 exhibited an unusually large Stokes shift (56 nm). An expanded set of genetically encoded Trp analogues should enable the design of new proteins with novel spectral properties.
Division of Chemistry and Chemical Engineering and Joseph J. Jacobs Institute for Molecular Engineering for Medicine, California Institute of Technology, Pasadena, California 91125, USA.
Inchan
I
Kwon
David A
DA
Tirrell
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D010652:Phenylalanine-tRNA Ligase