Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli
Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli
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DOI：
10.1002/bit.20524
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发表时间：
2005-08-20
影响因子：
3.8
通讯作者：
Takagi, M
中科院分区：
文献类型：
--
作者：
Sandee, D;Tungpradabkul, S;Takagi, M
许多真核蛋白已在大肠杆菌中成功表达。然而，并不是每个基因都能在这个生物体中高效表达。大多数蛋白质，特别是那些具有多个二硫键的蛋白质，已被证明在大肠杆菌中形成不溶于水的蛋白质或包涵体。非活性形式的蛋白质需要体外复性步骤才能恢复生物功能。在本研究中，我们描述了通过共表达DSB蛋白并用介质添加剂增强的抗肝癌单链可变区(ScFv)的可溶性表达系统。结果表明，DsbABCD蛋白的过表达对Hep27scFv的可溶性产量有显著影响，可能促进了正确的折叠。在培养基中添加0.5M山梨醇，可获得可溶性ScFv的最佳表达条件。竞争酶联免疫吸附试验(ELISA)表明，本方法表达的可溶性单链抗体与完整抗体(Hep27单抗)识别的肝癌细胞株(HCC-S102)上的同一表位具有结合活性。在此，我们报道了一种利用添加山梨醇培养基添加剂的DSB共表达系统在大肠杆菌中可溶性表达ScFv的有效方法。该方法可能适用于具有多个二硫键的蛋白质的高产量可溶表达。(C)2005年威利期刊公司。
Many eukaryotic proteins have been produced successfully in Escherichia coli. However, not every gene can be expressed efficiently in this organism. Most proteins, especially those with multiple disulfide bonds, have been shown to form insoluble protein or inclusion body in E. coli. An inactive form of protein would require an in vitro refolding step to regain biological functions. In this study, we described the system for soluble expression of a single-chain variable fragment (scFv) against hepatocellular carcinoma (Hep27scFv) by coexpressing Dsb protein and enhancing with medium additives. The results revealed that overexpression of DsbABCD protein showed marked effect on the soluble production of Hep27scFv, presumably facilitating correct folding. The optimal condition for soluble scFv expression could be obtained by adding 0.5M sorbitol to the culture medium. The competitive enzyme-linked immunosorbent assay (ELISA) indicated that soluble scFv expressed by our method retains binding activity toward the same epitope on a hepatocellular carcinoma cell line (HCC-S102) recognized by intact antibody (Ab) (Hep27 Mab). Here, we report an effective method for soluble expression of scFv in E. coli by the Dsb coexpression system with the addition of sorbitol medium additive. This method might be applicable for high-yield soluble expression of proteins with multiple disulfide bonds. (c) 2005 Wiley Periodicals, Inc.