期刊
JOURNAL OF BIOTECHNOLOGY
卷 145, 期 2, 页码 93-98出版社
ELSEVIER
DOI: 10.1016/j.jbiotec.2009.10.010
关键词
Surface display; Pichia pastoris; Nanobody; Camelid heavy chain antibody
资金
- Marie Curie Excellence Grant
- DWTC
Yeast surface display is an efficient tool for isolating and engineering antibody fragments, both scFv and Fab. We describe the use of protein display on Pichia pastoris for the rapid selection of camelid antibodies composed only of heavy chains (nanobodies) from a library derived from a Ilama immunized with Green Fluorescent Protein. The library of nanobody-coding sequences was fused to the C-terminal part of the Saccharomyces cerevisiae alpha-agglutinin gene (SAG1) and expressed in glycoengineered P. pastoris. A high efficiency transformation protocol yielded a library of 5 x 10(7) clones. About 80% of the clones strongly expressed the nanobody fusion. Nanobody-displaying clones were rapidly enriched by fluorescence activated cell sorting (FACS), and GFP-specific nanobody-displaying clones were isolated and equilibrium dissociation constants (K-d) determined. This technology for displaying protein libraries on P. pastoris enables the isolation and engineering of antibody-derived molecules in a robust eukaryotic expression host. (C) 2009 Elsevier B.V. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据