Cerezo Julieta, Smith María Emilia, Rodríguez Talou Julián
Instituto NANOBIOTEC - Cátedra de Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires - Consejo Nacional de Investigaciones Científicas y Técnicas, Junín 956, 1113, Ciudad Autónoma de Buenos Aires, Argentina.
Protein Expr Purif. 2019 Jan;153:131-137. doi: 10.1016/j.pep.2018.09.009. Epub 2018 Sep 18.
This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed. The protein was produced and purified directly from the yeast culture supernatant both efficiently and with no loss. The purified protein was properly immobilized by adsorption in solid phase and recognized by anti-dengue antibodies, showing its potential for the development of an indirect immunoassay for dengue virus.
这项工作描述了一种在毕赤酵母培养物中进行重组蛋白整合表达和纯化的新策略。疏水蛋白可用作融合标签，与其融合的蛋白质会改变其疏水性，并且可以通过基于非离子表面活性剂的双水相系统（ATPS）进行纯化。在此，将与里氏木霉疏水蛋白I融合的登革病毒包膜蛋白结构域III共识序列在毕赤酵母培养物中表达，并使用非离子洗涤剂（Triton X-114）通过双水相系统进行原位产物去除。该蛋白质直接从酵母培养上清液中高效生产和纯化，且无损失。纯化后的蛋白质通过吸附正确固定在固相上，并被抗登革热抗体识别，显示出其在开发登革病毒间接免疫测定中的潜力。
