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A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries.
Molecular Biotechnology ( IF 2.6 ) Pub Date : 2020-01-01 , DOI: 10.1007/s12033-019-00224-z
Davide Ferrari 1, 2 , Valentina Garrapa 1 , Massimo Locatelli 2 , Angelo Bolchi 1, 3
Affiliation  

Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 1012 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.

中文翻译:

优化细菌表达并适合构建核糖体展示文库的新型纳米抗体支架。

骆驼科抗体的单域抗原结合片段,称为VHH或纳米抗体,是广泛使用的亲和试剂。然而,它们涉及动物免疫的生产是时间和资源密集的。从美洲驼VHHs的序列数据集开始,我们基于保守的框架序列设计了一种新型支架,适用于细菌纳米抗体的表达和合成文库的构建。通过移植来自两个已知纳米抗体的CDR验证了共有支架。与相应的野生型相比,这两个嵌合纳米抗体在保持其结合特性的同时,在大肠杆菌中表现出更高的表达水平和溶解度。概念验证的合成组合库,适用于核糖体展示(RD)选择,通过在共有框架内编码三个随机互补决定区获得。该库由线性DNA片段组成,估计的复杂度> 1012,比普通噬菌体展示库高三个数量级。几个文库克隆的细菌表达表明高产量的可溶性重组蛋白。文库的高度复杂性通过克隆子集的测序以及麦芽糖结合蛋白结合剂的初步RD选择得到证实,表明该方法是构建合成组合文库用作动物实验的起点。免费工具,可低成本选择目标特定的纳米抗体。1012比普通噬菌体展示库高三个数量级。几个文库克隆的细菌表达表明高产量的可溶性重组蛋白。文库的高度复杂性通过克隆子集的测序以及麦芽糖结合蛋白结合剂的初步RD选择得到证实,表明该方法是构建合成组合文库用作动物实验的起点。免费工具,可低成本选择目标特定的纳米抗体。1012比普通噬菌体展示库高三个数量级。几个文库克隆的细菌表达表明高产量的可溶性重组蛋白。文库的高度复杂性通过克隆子集的测序以及麦芽糖结合蛋白结合剂的初步RD选择得到证实,表明该方法是构建合成组合文库用作动物实验的起点。免费工具,可低成本选择目标特定的纳米抗体。
更新日期:2019-11-01
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