High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

TitleHigh-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.
Publication TypeJournal Article
Year of Publication1994
AuthorsLaroche, Y., V. Storme, J. De Meutter, J. Messens, and M. Lauwereys
JournalBiotechnology (N Y)
Volume12
Issue11
Pagination1119-24
Date Published1994 Nov
ISSN0733-222X
KeywordsAmino Acid Sequence, Animals, Arthropod Proteins, Base Sequence, Carbon Isotopes, Cloning, Molecular, Factor Xa Inhibitors, Fermentation, Genetic Vectors, Molecular Sequence Data, Nitrogen Isotopes, Peptide Biosynthesis, Peptides, Pichia, Recombinant Proteins, Selection, Genetic, Ticks
Abstract

Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

Alternate JournalBiotechnology (N.Y.)
PubMed ID7765555
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