Residue-specific global fluorination of Candida antarctica lipase B in Pichia pastoris.

We report the in vivo fluorination of the tryptophan, tyrosine, and phenylalanine residues in a glycosylation-deficient mutant of Candida antarctica lipase B, CalB N74D, expressed in the methylotrophic yeast Pichia pastoris and subsequently segregated into the growth medium. To achieve this, a P. pastoris strain auxotrophic for all three aromatic amino acids was supplemented with 5-fluoro-L-tryptophan, meta-fluoro-(DL)-tyrosine, or para-fluoro-L-phenylalanine during expression of CalB N74D.

Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues.

We replaced the single N-terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non-canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae. Our non-canonical amino acid incorporation protocol involves a two-step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off.

Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code.

The expanded genetic code in combination with site-directed mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange ('atomic mutation') and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties.