Breast cancer is the second most commonly diagnosed cancer, worldwide. Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is correlated with poor prognosis. HER2-targeting monoclonal antibodies resulted in longer survival of HER2+ breast cancer. Single-chain variable fragment (scFv) demonstrates improved penetrability into tumors. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause formation of inclusion bodies. Disulfide bond can be formed properly in cytoplasm of SHuffle® strain as it is trxB-, gor-, and overexpresses cytoplasmic DsbC chaperone. In this study, the anti-HER2 scFv was successfully expressed and purified in BL21 (DE3) and SHuffle® cells. Here, significant higher soluble anti-HER2 scFv was produced in SHuffle® than in BL21 strain. The specific binding of anti-HER2 scFv to HER2 was shown by flow cytometry analysis and ELISA. Moreover, it was demonstrated that the anti-HER2 scFv produced in SHuffle® binds to HER2 at higher level as compared to that expressed in BL21 cells. Furthermore, competitive ELISA-based study suggested that anti-HER2 scFv recognizes the same epitope of HER2 receptor as the trastuzumab antibody. Our findings indicated that correct disulfide bond formation in SHuffle® strain can result in enhanced solubility and higher biological activity level of anti-HER2 scFv.

中文翻译：

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大肠杆菌SHuffle®T7Express细胞中的抗HER2 scFv表达：对溶解度和生物活性的影响。

乳腺癌是全球第二大最常被诊断出的癌症。人表皮生长因子受体2（HER2）过度表达的乳腺癌与不良预后相关。靶向HER2的单克隆抗体可导致HER2 +乳腺癌的更长生存期。单链可变片段（scFv）表现出提高的对肿瘤的渗透性。由于存在两个二硫键，scFv在减少细菌细胞质中的表达可能导致包涵体的形成。可以在SHuffle®菌株的细胞质中正确形成二硫键，因为它是trxB-，gor-和过表达细胞质DsbC分子伴侣。在这项研究中，抗HER2 scFv在BL21（DE3）和SHuffle®细胞中成功表达和纯化。在这里，SHuffle®产生的可溶性抗HER2 scFv明显高于BL21菌株。通过流式细胞术分析和ELISA显示抗HER2scFv与HER2的特异性结合。此外，已证明与在BL21细胞中表达的抗HER2 scFv相比，在SHuffle®中产生的抗HER2 scFv以更高的水平结合于HER2。此外，基于竞争性ELISA的研究表明抗HER2 scFv识别与曲妥珠单抗抗体相同的HER2受体表位。我们的发现表明，在SHuffle®菌株中正确的二硫键形成可导致抗HER2 scFv的溶解度提高和生物学活性更高。基于竞争性ELISA的研究表明，抗HER2 scFv识别与曲妥珠单抗抗体相同的HER2受体表位。我们的发现表明，在SHuffle®菌株中正确的二硫键形成可以导致抗HER2 scFv的溶解度提高和生物学活性更高。基于竞争性ELISA的研究表明，抗HER2 scFv识别与曲妥珠单抗抗体相同的HER2受体表位。我们的发现表明，在SHuffle®菌株中正确的二硫键形成可以导致抗HER2 scFv的溶解度提高和生物学活性更高。