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Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2017-08-14 00:00:00 , DOI: 10.1021/acssynbio.7b00144
Carlos A. Cruz-Teran 1 , Karthik Tiruthani 1 , Adam Mischler 1 , Balaji M. Rao 1
Affiliation  

The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by “self-cleaving” 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence—with ∼50% efficiency of ribosomal skipping—between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein–Aga2 fusion that is tethered to the yeast cell surface. We show that binding proteins derived from the Sso7d scaffold and the homodimeric enzyme glucose oxidase can be simultaneously secreted solubly and expressed as yeast cell surface fusions using the F2A-based system. Furthermore, a combinatorial library of Sso7d mutants can be screened to isolate binders with higher affinity for a model target (lysozyme), and the pool of higher affinity binders can be characterized in soluble form. Significantly, we show that both N- and C-terminal fusions to Aga2 can be simultaneously secreted solubly and displayed on the cell surface; this is particularly advantageous because protein functionality can be affected by the specific position of Aga2 in the protein fusion. We expect that the F2A-based yeast surface display and secretion system will be a useful tool for protein engineering and enable efficient characterization of individual clones isolated from combinatorial libraries.

中文翻译:

效率低下的核糖体跳跃使啤酒酵母中蛋白质的同时分泌和展示成为可能

对可溶性蛋白进行重组表达的需要减慢了从组合文库中分离的工程蛋白的验证速度,并限制了所评估蛋白变异体的数量。为了克服这个瓶颈,我们描述了一种同时用于细胞表面展示和酿酒酵母中蛋白质可溶性分泌的系统基于低效率的核糖体跳跃。“自我切割” 2A肽介导的核糖体跳跃从一个开放阅读框产生两种蛋白质。在目标蛋白和酵母细胞壁蛋白Aga2之间掺入F2A肽序列(效率约为50%的核糖体跳跃)可同时表达脱脂蛋白和拴系在酵母上的蛋白-Aga2融合蛋白细胞表面。我们表明,从Sso7d支架和同型二聚体酶葡萄糖氧化酶衍生的结合蛋白可以同时可溶地分泌,并使用基于F2A的系统表达为酵母细胞表面融合体。此外,可以筛选Sso7d突变体的组合文库,以分离对模型靶标(溶菌酶)具有更高亲和力的结合剂,高亲和力的粘合剂库可以以可溶形式表征。重要的是,我们显示与Aga2的N和C端融合都可以同时被可溶性分泌并显示在细胞表面。这是特别有利的,因为蛋白质功能性会受到蛋白质融合物中Aga2的特定位置的影响。我们期望基于F2A的酵母表面展示和分泌系统将成为蛋白质工程的有用工具,并能够有效地表征从组合文库中分离出的单个克隆。这是特别有利的,因为蛋白质功能性会受到蛋白质融合物中Aga2的特定位置的影响。我们期望基于F2A的酵母表面展示和分泌系统将成为蛋白质工程的有用工具,并使从组合文库中分离出的单个克隆的有效表征成为可能。这是特别有利的,因为蛋白质功能性会受到蛋白质融合物中Aga2的特定位置的影响。我们期望基于F2A的酵母表面展示和分泌系统将成为蛋白质工程的有用工具,并能够有效地表征从组合文库中分离出的单个克隆。
更新日期:2017-08-14
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