Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery.
Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery.
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DOI：
10.1016/j.jmb.2008.10.051
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发表时间：
2009-01-09
影响因子：
5.6
通讯作者：
DeLisa, Matthew P.
中科院分区：
文献类型：
--
作者：
Fisher, Adam C.;DeLisa, Matthew P.
最常用的重组抗体形式之一是单链可变片段（scFv），其由通过柔性接头连接至可变轻链的抗体可变重链组成。由于二硫键通常是scFv折叠所必需的，因此在胞质溶胶的还原环境中表达scFv可能具有挑战性。因此，我们试图开发一种用于在细菌的还原胞质溶胶中稳定的scFv的抗原非依赖性选择的方法。为此，我们应用了最近开发的遗传选择的基础上的大肠杆菌双精氨酸易位（达特）途径（蛋白质科学）的质量控制功能的蛋白质折叠和溶解度。该选择采用感兴趣的蛋白质与N-末端Tat特异性信号肽和C-末端TEM 1 β-内酰胺酶的三方夹心融合，从而将抗生素抗性与达特途径输出偶联。在此，我们调整该测定以开发达特输出后的胞内抗体选择（ISELATE），这是一种用于鉴定溶解度增强的scFv序列的高通量选择策略。使用ISELATE进行三轮实验室进化，可以从不溶性亲本序列进化出可溶性scFv。我们还表明，ISELATE能够在功能筛选之前将scFv文库集中在可溶性序列空间中，因此可以用于增加发现功能性胞内抗体的可能性。最后，该技术用于筛选大量的天然scFv，以获得赋予显著水平的可溶性积累的克隆。通过这些方法，我们表明达特质量控制机制可以用于可溶于E.杆菌
One of the most commonly used recombinant antibody formats is the single-chain variable fragment (scFv) that consists of the antibody variable heavy chain connected to the variable light chain by a flexible linker. Since disulfide bonds are often necessary for scFv folding, it can be challenging to express scFvs in the reducing environment of the cytosol. Thus, we sought to develop a method for antigen-independent selection of scFvs that are stable in the reducing cytosol of bacteria. To this end, we applied a recently developed genetic selection for protein folding and solubility based on the quality control feature of the Escherichia coli twin-arginine translocation (Tat) pathway ( Protein Sci). This selection employs a tripartite sandwich fusion of a protein-of-interest with an N-terminal Tat-specific signal peptide and C-terminal TEM1 β-lactamase, thereby coupling antibiotic resistance with Tat pathway export. Here, we adapted this assay to develop intrabody selection after Tat export (ISELATE), a high-throughput selection strategy for the identification of solubility-enhanced scFv sequences. Using ISELATE for three rounds of laboratory evolution, it was possible to evolve a soluble scFv from an insoluble parental sequence. We also show that ISELATE enables focusing of an scFv library in soluble sequence space prior to functional screening and thus can be used to increase the likelihood of finding functional intrabodies. Finally, the technique was used to screen a large repertoire of naïve scFvs for clones that conferred significant levels of soluble accumulation. In these ways, we show that the Tat quality control mechanism can be harnessed for molecular evolution of scFvs that are soluble in the reducing cytoplasm of E. coli.
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影响因子：
46.9
作者：
de Wildt, RMT;Mundy, CR;Tomlinson, IM
通讯作者：
Tomlinson, IM
DOI：
10.1073/pnas.95.24.14130
发表时间：
1998-11-24
影响因子：
11.1
作者：
Hanes, J;Jermutus, L;Plückthun, A
通讯作者：
Plückthun, A
影响因子：
46.9
作者：
Cabantous, S;Terwilliger, TC;Waldo, GS
通讯作者：
Waldo, GS
DOI：
10.1093/protein/11.12.1267
发表时间：
1998-12-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
de Haard, HJW;Kazemier, B;Hoogenboom, HR
通讯作者：
Hoogenboom, HR
影响因子：
8
作者：
Fisher, AC;Kim, W;DeLisa, MP
通讯作者：
DeLisa, MP