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Effects of Cultivation Conditions on the Expression Level of Recombinant scFv Antibody against EpEX in Escherichia coli

机译:培养条件对大肠杆菌中EpEX重组scFv抗体表达水平的影响

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The EpCAM (epithelial cell adhesion molecule) is a cell surface antigen over expressed in many types of epithelial cell cancers including colon, stomach, pancreas, lung, ovarian, and breast. So, it can be an attractive target for active and passive immunotherapy of cancers. ScFv (single chain fragment variable) fragment is a class of engineered antibodies in which the genes coding for the heavy (VH) and light chains (VL) of an immunoglobulin have been linked with a short flexible peptide linker. Inexpensive media, rapid growth rates, and relatively minimal laboratory set up make Escherichia coli (E. coli) a suitable host for expression of a large variety of recombinant proteins. Here, we assessed the effect of cultivation conditions on the level of expressed scFv against extracellular domain of EpCAM (EpEX) in E. coli. pET22b-antiEpEX-scFv was transformed into prepared E. coli Rosetta?(DE3) competent cells. To evaluate the effect of cultivation conditions on protein expression level, three factors of incubation temperature (25, 30, 37°C), the IPTG (isopropyl-β-D-thiogalactoside) concentration (0.1, 0.25, 0.5, 1 mM), and induction duration (3, 5, 7, 18 h) were considered. SDS-PAGE and western blot analysis demonstrated an estimated 30 kDa-size protein band which was related to the recombinant scFv expressed in E. coli Rosetta?(DE3) strain. At optimal condition (5 h after induction with 0.5 mM IPTG at 25 °C), the final production yield of the antiEpEX-scFv was 403.29 ± 87.50 μg/mL. Our results provide a foundation for the development of scFv-based drugs’ production as effective therapeutic agents in cancers with epithelial origin. HIGHLIGHTS The antiEpEX-scFv was successfully expressed in E. coli Rosetta?(DE3) strain. The highest concentration of protein was obtained with 0.5 mM IPTG at 30°C. The final yield of recombinant antiEpEX-scFv was approximately 403.29 ± 87.50 mg/L.
机译:EpCAM(上皮细胞粘附分子)是一种细胞表面抗原,在许多类型的上皮细胞癌中过度表达,包括结肠癌,胃癌,胰腺癌,肺癌,卵巢癌和乳腺癌。因此,它可以成为主动和被动癌症免疫疗法的诱人靶标。 ScFv(单链片段可变)片段是一类工程抗体,其中编码免疫球蛋白重链(VH)和轻链(VL)的基因已与一个短的柔性肽接头连接。廉价的培养基,快速的生长速度和相对较少的实验室设置使大肠杆菌(E. coli)成为表达多种重组蛋白的合适宿主。在这里,我们评估了培养条件对大肠杆菌中EpCAM(EpEX)胞外域表达的scFv水平的影响。将pET22b-antiEpEX-scFv转化为准备好的大肠杆菌Rosetta™(DE3)感受态细胞。为了评估培养条件对蛋白质表达水平的影响,孵育温度(25、30、37°C​​),IPTG(异丙基-β-D-硫代半乳糖苷)浓度(0.1、0.25、0.5、1 mM)的三个因素,并考虑诱导持续时间(3、5、7、18小时)。 SDS-PAGE和蛋白质印迹分析表明,估计有30kDa大小的蛋白条带,它与在大肠杆菌RosettaTM(DE3)菌株中表达的重组scFv有关。在最佳条件下(在25°C下用0.5 mM IPTG诱导5小时后),antiEpEX-scFv的最终产量为403.29±87.50μg/ mL。我们的结果为开发基于scFv的药物作为上皮来源的癌症的有效治疗剂奠定了基础。亮点antiEpEX-scFv在大肠杆菌Rosetta?(DE3)菌株中成功表达。在30°C下用0.5 mM IPTG可获得最高浓度的蛋白质。重组抗EpEX-scFv的最终产量约为403.29±87.50 mg / L。

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