Isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris
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- 作者：Stefan Ryckaert ; Els Pardon ; Jan Steyaert ; Nico Callewaert
- 关键词：Surface display ; Pichia pastoris ; Nanobody ; Camelid heavy chain antibody
- 刊名：Journal of Biotechnology
- 出版年：2010
- 出版时间：15 January 2010
- 年：2010
- 卷：145
- 期：2
- 页码：93-98
- 全文大小：651 K
文摘
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 llama 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 × 107 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 (Kd) 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.