Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, SE-106 91, Stockholm, Sweden.
Biotechnol J. 2019 Apr;14(4):e1800359. doi: 10.1002/biot.201800359. Epub 2018 Sep 25.
Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naïve affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10 variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.
利用组合蛋白工程开发新的亲和蛋白，这已经成为生成单克隆抗体的既定方法，对于发现基于非免疫球蛋白蛋白的结合物也至关重要。噬菌体展示技术是最常用的方法，但酵母展示技术也越来越流行，部分原因是可以利用荧光激活细胞分选（FACS）来分离新的候选物。大肠杆菌具有用于文库应用的多种有价值的特性，特别是高转化效率。各种自转运蛋白和 intimins 用于在外膜上的分泌和锚定已经显示出有希望的结果，特别是对于不同酶的定向进化。在这里，作者报告了使用自转运蛋白 Adhesin Involved in Diffuse Adherence (AIDA-I) 在大肠杆菌外膜上展示大型天然亲和体文库。表达盒首先通过去除非必需序列进行工程设计，然后将包含 10 多种变体的亲和体文库引入新的展示载体。通过 FACS 针对五个不同的靶标评估文库的质量和方法的一般性能，结果产生了一组具有低纳摩尔亲和力的结合物，表明该方法有可能作为噬菌体展示的补充，用于生成亲和体分子。
