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 (tape) based on codons preferentially obs erved 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 wa s correctly processed at the amino-terminal fusion site, and accumulat ed in the medium to: approximately 1.7 g/l. This corresponds to a mola r concentration of 0.24 mM, and is the highest yet described for a rec ombinant product secreted from P. pastoris. It also represents a seven -fold improvement in productivity compared to rTAP secretion from Sacc haromyces cerevisiae, making P. pastoris an attractive host for the in dustrial-scale production of this potential therapeutic agent. This sy stem was also used to prepare 21 mg N-15-rTAP, 11 mg C-13-rTAP and 27 mg N-15/C-13-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.