Mani Sheida, Arab Bahareh, Akbari Vajihe, Chou C Perry
Pharmacy Student Research Committee, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Chemical Engineering, University of Waterloo, 200 University Avenue, Waterloo, ON, N2L 3G1, Canada.
AMB Express. 2024 Sep 28;14(1):107. doi: 10.1186/s13568-024-01765-6.
Immunotoxins are widely applied for cancer therapy. However, bacterial expression of immunotoxins usually leads to the formation of insoluble and non-functional recombinant proteins. This study was aimed to improve soluble expression of a novel anti-HER2 immunotoxin under the regulation of the trc promoter in Escherichia coli by optimization of the cultivation conditions using response surface methodology (RSM). To conduct RSM, four cultivation variables (i.e., inducer concentration, post-induction time, post-induction temperature, and medium recipe), were selected for statistical characterization and optimization using the Box-Behnken design and Design Expert software. Based on the developed model using the Box-Behnken design, the optimal cultivation conditions for soluble expression of anti-HER2 immunotoxin were determined to be 0.1 mM IPTG for induction in the LB medium at 33 °C for 18 h. The expressed immunotoxin was successfully purified using affinity chromatography with more than 90% purity and its bioactivity was confirmed using cell-based ELISA. Technical approach developed in this study can be generally applied to enhance the production yield and quality of recombinant proteins using E. coli as the gene expression system.
免疫毒素被广泛应用于癌症治疗。然而，免疫毒素在细菌中的表达通常会导致不溶性和无功能的重组蛋白形成。本研究旨在通过使用响应面法（RSM）优化培养条件，提高新型抗HER2免疫毒素在大肠杆菌trc启动子调控下的可溶性表达。为了进行RSM，选择了四个培养变量（即诱导剂浓度、诱导后时间、诱导后温度和培养基配方），使用Box-Behnken设计和Design Expert软件进行统计表征和优化。基于使用Box-Behnken设计开发的模型，确定抗HER2免疫毒素可溶性表达的最佳培养条件为在LB培养基中于33℃用0.1 mM IPTG诱导18小时。使用亲和色谱成功纯化了表达的免疫毒素，纯度超过90%，并使用基于细胞的ELISA证实了其生物活性。本研究中开发的技术方法通常可用于提高以大肠杆菌作为基因表达系统的重组蛋白的产量和质量。
