High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.
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.
Laroche
Y
Y
Corvas International N.V., Gent, Belgium.
Storme
V
V
De Meutter
J
J
Messens
J
J
Lauwereys
M
M
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D002247:Carbon Isotopes; D003001:Cloning, Molecular; D065427:Factor Xa Inhibitors; D005285:Fermentation; D005822:Genetic Vectors; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics*; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012641:Selection, Genetic; D013987:Ticks
D060829:Arthropod Proteins; D002247:Carbon Isotopes; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D009587:Nitrogen Isotopes; D010455:Peptides; D011994:Recombinant Proteins; C063967:tick anticoagulant peptide