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首页> 外文期刊>Biological chemistry >Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system.
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Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system.

机译：在原核无细胞表达系统中功能完整的IgG的合成和表征。

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摘要

Abstract Antibodies are an important component of the immune system of higher eukaryotes. Furthermore, they are effective tools in basic research, medical diagnostics and therapy. Recombinant expression of these heterotetrameric, disulfide-bridged proteins is usually performed in mammalian cells. Here, we describe the cell-free expression of a mouse monoclonal antibody, MAK33, in a coupled transcription/translation system, based on an Escherichia coli lysate. Both the heavy and the light chain can be produced efficiently in this setup. However, they fail to form functional antibodies. With a view to overcome folding and oxidation defects, we supplemented the system with the oxidoreductases PDI (protein disulfide isomerase) and DsbC and the ER-specific chaperones Grp94 and BiP; furthermore, we optimized the redox conditions. We found that functional antibodies can only be obtained in the presence of an oxidoreductase. In contrast, the addition of Grp94 and/or BiP had no influence on the productive folding reaction. The comparison of the antibody expressed in vitro with MAK33 expressed in cell culture showed that the in vitro expressed antibody is correctly assembled, disulfide-bridged and shows identical antigen affinity. The stability of the in vitro expressed non-glycosylated IgG is comparable to that of the authentic antibody.
机译：摘要抗体是高等真核生物免疫系统的重要组成部分。此外，它们是基础研究，医学诊断和治疗中的有效工具。这些异四聚体，二硫键桥接的蛋白质的重组表达通常在哺乳动物细胞中进行。在这里，我们描述了基于大肠杆菌溶菌产物的小鼠单克隆抗体MAK33在偶联转录/翻译系统中的无细胞表达。在这种设置下，重链和轻链都可以高效生产。但是，它们不能形成功能性抗体。为了克服折叠和氧化缺陷，我们在系统中添加了氧化还原酶PDI（蛋白二硫键异构酶）和DsbC以及ER特异性伴侣蛋白Grp94和BiP。此外，我们优化了氧化还原条件。我们发现功能性抗体只能在氧化还原酶存在下获得。相反，添加Grp94和/或BiP对生产性折叠反应没有影响。体外表达的抗体与细胞培养物中表达的MAK33的比较表明，体外表达的抗体正确组装，二硫键桥接并显示出相同的抗原亲和力。体外表达的非糖基化IgG的稳定性与真实抗体相当。

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