Laroche Y, Storme V, De Meutter J, Messens J, Lauwereys M
Corvas International N.V., Gent, Belgium.
Biotechnology (N Y). 1994 Nov;12(11):1119-24. doi: 10.1038/nbt1194-1119.
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.
蜱抗凝肽（TAP）是一种强效且特异性的凝血蛋白酶因子Xa抑制剂。我们基于在高表达的毕赤酵母醇氧化酶1基因（AOX1）中优先观察到的密码子，设计并组装了一个合成的TAP编码基因（tapo），并将其与一个新型的杂交分泌前导肽序列融合。该基因的表达产生了具有生物活性的重组TAP（rTAP），它在氨基末端融合位点被正确加工，并在培养基中积累至约1.7 g/L。这对应于0.24 mM的摩尔浓度，是迄今报道的毕赤酵母分泌的重组产物中最高的浓度。与酿酒酵母分泌rTAP相比，其生产力提高了7倍，这使得毕赤酵母成为工业规模生产这种潜在治疗剂的有吸引力的宿主。该系统还用于制备21 mg 15N-rTAP、11 mg 13C-rTAP和27 mg 15N/13C-rTAP，同位素掺入水平高于98%，纯度足以用于使用高场核磁共振确定蜱抗凝肽的溶液结构。
