Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.
Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.
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工程细菌细胞质中全长抗体的有效表达。
DOI：
10.1038/ncomms9072
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发表时间：
2015-08-27
影响因子：
16.6
通讯作者：
Berkmen M
中科院分区：
文献类型：
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作者：
Robinson MP;Ke N;Lobstein J;Peterson C;Szkodny A;Mansell TJ;Tuckey C;Riggs PD;Colussi PA;Noren CJ;Taron CH;DeLisa MP;Berkmen M
目前在工程细胞中生产免疫球蛋白G（IgG）抗体的方法通常需要重新折叠步骤或跨一个或多个生物膜分泌。在这里，我们描述了一个强大的表达平台，用于在大肠杆菌细胞质中生物合成全长IgG抗体。合成的重链和轻链都缺乏典型的输出信号，在特别工程化的E.允许在细胞质内形成稳定二硫键的大肠杆菌菌株。具有临床相关抗原和效应子结合活性的IgG在大肠杆菌中容易产生。通过将抗原特异性可变重链和轻链结构域移植到胞质稳定的框架中，并用促进与Fcγ受体结合的氨基酸取代重塑片段可结晶结构域，由此产生的细胞质IgG-命名为“cyclonals”-有效地绕过膜易位和糖基化的潜在限速步骤。 目前生产单克隆抗体的方法通常需要重折叠步骤或跨生物膜分泌。在这里，罗宾逊等人描述了工程E.用于在细菌细胞质中有效生产功能性免疫球蛋白G抗体的大肠杆菌菌株。
Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. Current methods for production of monoclonal antibodies often require refolding steps or secretion across biological membranes. Here, Robinson et al. describe engineered E. coli strains for efficient production of functional immunoglobulin G antibodies in the bacterial cytoplasm.