Centro de Investigación y Asistencia en Tecnología y diseño del Estado de Jalisco A.C., Av. Normalistas 800, C.P. 44270 Guadalajara, Mexico.
Centro Universitario de Los Altos, Universidad de Guadalajara, Km 7.5 Carretera a Yahualica, CP 47600 Tepatitlán de Morelos, Mexico.
Int J Biol Macromol. 2021 Feb 28;171:82-88. doi: 10.1016/j.ijbiomac.2020.12.179. Epub 2021 Jan 6.
Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 μg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes.
翻译工程和生物信息学加快了基因序列改进的速度，从而产生了多表位蛋白。用于结核病诊断的强抗原蛋白包括单独的 ESAT6 和 CFP10 蛋白或衍生肽。在不形成包含体的情况下在大肠杆菌中获得异源多组分抗原仍然是一项生物技术挑战。ESAT6-CFP10 融合抗原的基因序列经过密码子偏爱性调整，以高水平表达可溶性蛋白。通过 Ni 亲和层析获得的 23.7 kDa 的融合蛋白在 SDS-PAGE 和 Western blot 分析中观察到，在 IPTG 诱导后摇瓶培养中表达的可溶性蛋白的产量达到约 67 mg/L 的浓度。在牛的全血培养物中以 4 μg/mL 的浓度评估抗原性，并使用特异性 IFN-γ 释放测定体外评估蛋白质刺激。该杂交蛋白能够刺激牛结核病疑似病例的 T 细胞特异性反应。结果表明，改进的大肠杆菌密码子用法是一种很好且具有成本效益的策略，可以大规模生产具有持续抗原特性的多表位蛋白，用于诊断目的。
