Expression and purification of a broad-spectrum human protease inhibitor in Pichia pastoris

IF 2.3 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Varsha Bhakta , Negin Chaeichi Tehrani , Antje Ask , William P. Sheffield
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引用次数: 0

Abstract

Alpha-1 antitrypsin (AAT) is the most abundant member of the serpin superfamily of protease inhibitors found in human plasma. Expression of a broad-spectrum AAT variant (AAT M358R) and an AAT variant (AAT-RC-2) that specifically inhibits coagulation Factor XIa (FXIa) in the methylotrophic yeast Pichia pastoris were compared. When protein secretion was directed by the 85 amino acid Saccharomyces cerevisiae alpha mating factor (AMF) prepro signal sequence 1 (ss1), AAT-RC-2 was purified as a 45 kDa homogeneous polypeptide preparation, but AAT M358R was heterogeneous. Replacement of the ss1 prepro sequence with any of 5 presequences lacking pro sequences eliminated the heterogeneity. The highest yield was obtained with ss3-AAT M358R, where ss3 was the 19 amino acid Saccharomyces cerevisiae AMF presequence. Enzymatic deglycosylation of ss1-AAT M358R converted its high molecular weight heterogeneity into a simple combination of 55 kDa and 45 kDa polypeptides consistent with failure to remove the AMF presequence and inhibition of the Kex2 propeptide convertase by AAT M358R. Purified ss3-AAT M358R inhibited FXIa significantly more rapidly than E. coli-derived AAT M358R with indistinguishable reaction stoichiometry; purified ss1-AAT-RC-2 did not differ from its E. coli-derived counterpart in either kinetic parameter. Both AAT variants formed denaturation-resistant complexes with FXIa. Substitution of the AMF prepro sequence with its constituent presequence eliminated the protein expression problem caused by inhibition of the Pichia pastoris propeptide processing machinery by AAT M358R and will make feasible comparison of AAT M358R and AAT-RC-2 in animal models of thrombosis and bleeding.
广谱人蛋白酶抑制剂在毕赤酵母中的表达与纯化
α -1抗胰蛋白酶(AAT)是在人血浆中发现的蛋白酶抑制剂蛇形蛋白超家族中最丰富的成员。比较了一种广谱AAT变体(AAT M358R)和一种特异性抑制凝血因子XIa (FXIa)的AAT变体(AAT- rc -2)在甲基营养酵母毕赤酵母中的表达。在85个氨基酸的酿酒酵母α交配因子(AMF)前信号序列1 (ss1)的指导下,AAT- rc -2被纯化为45 kDa的均质多肽,而AAT M358R则是异质多肽。将ss1 prepro序列替换为缺乏pro序列的5个prepro序列中的任何一个,消除了异质性。ss3- aat M358R的产率最高,其中ss3为19个氨基酸的酿酒酵母AMF序列。ss1-AAT M358R的酶解糖基化将其高分子量异质性转化为55 kDa和45 kDa多肽的简单组合,这与AAT M358R无法去除AMF前序和抑制Kex2前肽转化酶一致。纯化的ss3-AAT M358R对FXIa的抑制速度明显高于大肠杆菌衍生的AAT M358R,反应化学计量学难以区分;纯化的ss1-AAT-RC-2与大肠杆菌衍生的对应物在任何动力学参数上都没有差异。两种AAT变体均与FXIa形成抗变性复合物。AMF prepro序列与其组成前序列的替换消除了AAT M358R抑制毕赤酵母前肽加工机制所导致的蛋白表达问题,并使AAT M358R与AAT- src -2在血栓和出血动物模型中的比较成为可能。
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来源期刊
Biochemistry and Biophysics Reports
Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics
CiteScore
4.60
自引率
0.00%
发文量
191
审稿时长
59 days
期刊介绍: Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.
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