High–Level Secretion and Very Efficient Isotopic Labeling of Tick Anticoagulant Peptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris
High–Level Secretion and Very Efficient Isotopic Labeling of Tick Anticoagulant Peptide (TAP) Expressed in the Methylotrophic Yeast, Pichia pastoris
复制标题
甲基营养酵母毕赤酵母中表达的蜱抗凝血肽 (TAP) 的高水平分泌和非常有效的同位素标记
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发表时间：
1994
期刊：
影响因子：
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通讯作者：
M. Lauwereys
中科院分区：
文献类型：
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作者：
Y. Laroche;V. Storme;J. Meutter;J. Messens;M. Lauwereys
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.
