The improvement of anti-HER2 scFv soluble expression in Escherichia coli

The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2+ tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemicallysynthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 ºC. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 ºC).

人表皮生长因子受体2（HER2）的表达与乳腺肿瘤恶性程度的相关性，推动了靶向HER2阳性（HER2+）肿瘤的抗体研发。此外，单链可变区片段（single-chain variable fragment，scFv）作为抗体最小的抗原结合区域，其分子内包含两个二硫键，在大肠杆菌（Escherichia coli）中表达时，往往会在细胞质内积累无功能的蛋白。经氧化还原改造的大肠杆菌菌株如奥里加米(DE3)（Origami (DE3)），可促进细胞质内正确二硫键的形成。本研究旨在优化奥里加米(DE3)菌株中抗HER2 scFv的表达，并探究诱导温度与宿主菌株对该蛋白可溶性的影响。为此，我们将化学合成的曲妥珠单抗（Trastuzumab）抗HER2 scFv克隆至pET-22b(+)载体中。结果显示，抗HER2 scFv可在奥里加米(DE3)菌株中成功表达，经Ni-NTA亲和柱纯化后，通过抗His抗体进行蛋白质免疫印迹（Western blot）分析可实现有效检测。在37℃条件下以1 mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导24小时，奥里加米(DE3)菌株中抗HER2 scFv的表达量达到峰值。进一步研究发现，与奥里加米(DE3)菌株相比，BL21菌株的抗HER2 scFv总表达水平更高。但奥里加米(DE3)菌株中抗HER2 scFv的可溶/不可溶形式比例有所提升。此外，当重组奥里加米(DE3)菌株在较低温度（25℃）下培养时，可获得更高的可溶性蛋白表达量。