使用GlycoSwitch质粒修饰N-糖基化途径以产生均一的、类人聚糖。
Modification of the N-glycosylation pathway to produce homogeneous, human-like glycans using GlycoSwitch plasmids.
作者信息
Vervecken Wouter, Callewaert Nico, Kaigorodov Vladimir, Geysens Steven, Contreras Roland
机构信息
Department of Molecular Biomedical Research, Ghent University, Ghent, Belgium.
出版信息
Methods Mol Biol. 2007;389:119-38. doi: 10.1007/978-1-59745-456-8_9.
Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.
糖基化是治疗性应用中异源蛋白生产的一个重要问题。在毕赤酵母中产生的糖蛋白含有高甘露糖聚糖结构，这可能会妨碍下游加工，可能具有免疫原性，并导致其从循环中快速清除。本章描述了一种通过使OCH1失活、过表达带有HDEL标签的甘露糖苷酶以及过表达Kre2/GlcNAc-转移酶I嵌合酶来帮助解决这些与糖基化相关问题的方法。文中还介绍了不同的质粒以及聚糖分析方法。
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