title: Residue-specific global fluorination of Candida antarctica lipase B in Pichia pastoris.
abstract: 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. The residue-specific replacement of the canonical amino acids by their fluorinated analogs was confirmed by mass analysis. Although global fluorination induced moderate changes in the secondary structure of CalB N74D, the fluorous variant proteins were still active lipases. However, their catalytic activity was lower than that of the non-fluorinated parent protein while their resistance to proteolytic degradation by proteinase K remained unchanged. Importantly, we observed that the global fluorination prolonged the shelf life of the lipase activity, which is an especially useful feature for the storage of, e.g., therapeutic proteins. Our study represents the first step on the road to the production of biotechnologically and pharmacologically relevant fluorous proteins in P. pastoris.
authors.affiliation: Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
authors.forename: Waltraud
authors.initials: W
authors.lastname: Wenger
authors.forename: Birgit
authors.initials: B
authors.lastname: Wiltschi
mesh_terms: D002853:Chromatography, Liquid; D002942:Circular Dichroism; D005656:Fungal Proteins; D054879:Halogenation / Q000502:physiology; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica