GlycoFi Inc., A wholly-owned subsidiary of Merck & Co Inc., Lebanon, NH 03766, United States.
J Immunol Methods. 2012 Jan 31;375(1-2):159-65. doi: 10.1016/j.jim.2011.10.003. Epub 2011 Oct 12.
A fragment of antigen binding (Fab) surface display system was developed using a glycoengineered Pichia pastoris host strain genetically modified to secrete glycoproteins with mammalian mannose-type Man(5)GlcNAc(2) N-linked glycans. The surface display method described here takes advantage of a pair of coiled-coil peptides as the linker while using the Saccharomyces cerevisiae Sed1p GPI-anchored cell surface protein as an anchoring domain. Several Fabs were successfully displayed on the cell surface using this system and the expression level of the displayed Fabs was correlated to that of secreted Fabs from the same glycoengineered host in the absence of the cell wall anchor. Strains displaying different model Fabs were mixed and, through cell sorting, the strain displaying more expressed Fab molecule or the strain displaying the Fab with higher affinity for an antigen was effectively enriched by FACS. This novel yeast surface display system provides a general platform for the display of Fab libraries for affinity and/or expression maturation using glycoengineered Pichia.
使用经过糖基工程改造的毕赤酵母宿主菌株，该菌株能够分泌具有哺乳动物甘露糖型 Man(5)GlcNAc(2) N-连接聚糖的糖蛋白，开发了抗原结合（Fab）表面展示系统的片段。这里描述的表面展示方法利用一对卷曲螺旋肽作为接头，同时使用酿酒酵母 Sed1p GPI-锚定细胞表面蛋白作为锚定结构域。使用该系统成功地在细胞表面展示了几种 Fab，并且展示的 Fab 的表达水平与相同糖基工程化宿主中无细胞壁锚定的分泌 Fab 的表达水平相关。展示不同模型 Fab 的菌株被混合，通过细胞分选，通过 FACS 有效地富集表达更多 Fab 分子的菌株或对抗原具有更高亲和力的 Fab 展示菌株。这种新型酵母表面展示系统为使用糖基工程化毕赤酵母展示 Fab 文库的亲和力和/或表达成熟提供了一个通用平台。
