A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris.
A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris.
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一种组合遗传文库方法，将异源糖基化酶靶向巴斯德毕赤酵母的内质网或高尔基体。
DOI：
10.1002/yea.1835
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Gerngross,TillmanU
中科院分区：
文献类型：
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作者：
Nett,JuergenH;Stadheim,TerranceA;Li,Huijuan;Bobrowicz,Piotr;Hamilton,StephenR;Davidson,RobertC;Choi,Byung-Kwon;Mitchell,Teresa;Bobrowicz,Beata;Rittenhour,Alissa;Wildt,Stefan;Gerngross,TillmanU
为了使酵母Pichia pastoris中的糖基化途径人源化，我们开发了几种组合基因文库，并使用它们来正确定位活性真核甘露聚糖酶和糖转移酶。在这里，我们报告了真菌II型膜蛋白的多达66个N末端靶向序列与异源糖基化酶的33个催化结构域融合的细节。我们表明，虽然很难预测哪一个前导/催化结构域将导致所需的活性，但融合蛋白文库的分析允许选择功能正常的前导/催化结构域组合。这种组合方法与高通量筛选方案一起，使我们能够人源化酵母糖基化途径，以分泌具有复杂N-糖基化的人糖蛋白。版权所有© 2011约翰威利父子有限公司.
To humanize the glycosylation pathway in the yeastPichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66N‐terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high‐throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complexN‐glycosylation. Copyright © 2011 John Wiley & Sons, Ltd.