Issue 9, 2010
From the journal:

Molecular BioSystems

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

Nediljko Budisa,a   Waltraud Wengera  and  Birgit Wiltschi*abc  

* Corresponding authors

a Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

b Bioss–Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Engesserstrasse 4b, 79108 Freiburg, Germany
E-mail: birgit.wiltschi@biologie.uni-freiburg.de
Fax: +49 761 203 97660
Tel: +49 761 203 97656

c Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany

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.

Graphical abstract: Residue-specific global fluorination of Candida antarctica lipase B in Pichia pastoris

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Article information

DOI
https://doi.org/10.1039/C002256J
Article type
Paper
Submitted
02 Feb 2010
Accepted
16 Mar 2010
First published
29 Apr 2010

Mol. BioSyst., 2010,6, 1630-1639

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

N. Budisa, W. Wenger and B. Wiltschi, Mol. BioSyst., 2010, 6, 1630 DOI: 10.1039/C002256J

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