Protein Engineering via Site-Specific Incorporation of Nonnatural Amino Acids

Citation

Kwon, Inchan (2007) Protein Engineering via Site-Specific Incorporation of Nonnatural Amino Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/XVPN-9P37. https://resolver.caltech.edu/CaltechETD:etd-01222007-010333

Abstract

Nonnatural amino acid incorporation has been one of the most important protein engineering techniques. Particularly site-specific incorporation of nonnatural amino acids would allow design of artificial proteins containing a nonnatural amino acid with minimal perturbation of their native properties. Site-specific incorporation of phenylalanine (Phe) analogs and tryptophan (Trp) analogs, such as p-bromophenylalanine (pBrF), p-iodophenylalanine, p-azidophenylalanine, 6-chlorotrytophan, 6-bromotryptophan, 5-bromotrytophan, and benzothienylalanine, into proteins in Escherichia coli has been realized by E. coli strains outfitted with a mutant yeast phenylalanyl-tRNA suppressor (ytRNA^Phe_CUA) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) with a point mutation in the active site of the enzyme. In order to reduce Trp and lysine (Lys) misincorporation at an amber codon, the ytRNA^Phe_{CUA_UG} containing the optimized sequence and the yPheRS (T415A) showing higher specificity toward pBrF were developed. Combining ytRNA^Phe_{CUA_UG} and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.

Re-assignment of degenerate sense codons offers the prospect of a substantially expanded genetic code and a correspondingly enriched set of building blocks for natural and artificial proteins. Here we describe the use of a mutant yeast phenylalanine transfer RNA (ytRNA^Phe_AAA) containing a modified anticodon to break the degeneracy of the genetic code in E. coli. By using an E. coli strain co-transformed with ytRNA^Phe_AAA and a mutant yPheRS (T415G), we demonstrated efficient replacement of Phe by L-3-(2-naphthyl)alanine (2Nal) at UUU, but not at UUC codons.

However, this method had two limitations. First, the yPheRS (T415G) also activated Trp, which led to Trp misincorporation. Second, 2Nal was misincorporated at UUC Phe codons, due to the relaxed codon recognition of AAA anticodon in the ytRNA^Phe_AAA. High-throughput screening of a yPheRS library led to a more selective yPheRS variant (yPheRS_naph). The rationally designed ytRNA^Phe_CAA, which has the CAA anticodon recognizing only a UUG (Leu) codon, allowed incorporation of 2Nal only at UUG codon. Combined use of yPheRS_naph and ytRNA^Phe_CAA achieved multiple-site-specific incorporation of 2Nal into proteins. These results illustrate a general method for increasing the number of distinct, genetically-encoded amino acids available for protein engineering.

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