干扰素-β (IFN-β) 是一种具有广泛生物和制药应用的细胞因子，包括多发性硬化症 (MS)、癌症、一些自身免疫性疾病和病毒感染性疾病。因此，已经进行了许多研究来开发新的策略，以经济有效的方法高产地生产功能性 IFN-β。本研究旨在提高毕赤酵母中IFN-β-1a的胞内表达。将IFN-β-1a基因成功亚克隆到pPICZA载体中。通过电穿孔将重组载体转染至毕赤酵母GS115细胞。筛选阳性毕赤酵母转化子后，评估IFN-β-1a的表达，并优化培养条件，包括温度、培养时间和甲醇浓度。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析蛋白表达水平。EcoRI和XhoI限制性内切酶双酶切和序列分析证实IFN-β-1a基因正确亚克隆到pPICZA中。 SDS-PAGE分析表明，2%甲醇在28°C孵育72小时后，产生最高水平的IFN-β-1a（每1L酵母培养物25mg）。优化细胞内表达IFN-的培养条件β-1a 实验顺利进行。这种方法通常可用于提高巴斯德毕赤酵母中其他重组蛋白的产量和质量。版权所有：© 2024 Advanced Biomedical Research。

Interferon-beta (IFN-β) is a cytokine with a wide range of biological and pharmaceutical applications, including multiple sclerosis (MS), cancer, some autoimmune disorders, and viral infectious diseases. Thus, many studies have been performed to develop novel strategies for the high-yield production of functional IFN-β in a cost-effective approach. Here, we aimed to improve the intracellular expression of IFN-β-1a in Pichia pastoris.The gene of IFN-β-1a was successfully sub-cloned into the pPICZA vector. The recombinant vector was transfected to P. pastoris GS115 cells by electroporation. After screening positive P. pastoris transformants, the expression of IFN-β-1a was evaluated and the cultivation conditions, including temperature, time of incubation, and methanol concentration, were optimized. The protein expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The double digestion with EcoRI and XhoI restriction enzymes and sequence analysis confirmed the correct sub-cloning of the IFN-β-1a gene into pPICZA. SDS-PAGE analysis showed that the highest level of IFN-β-1a (25 mg per 1 L of yeast culture) was produced with 2% methanol at 28°C after 72 h incubation.Optimization of cultivation conditions for intracellular expression of IFN-β-1a was successfully performed. This approach can be generally applied to improve the production yield and quality of other recombinant proteins in P. pastoris.Copyright: © 2024 Advanced Biomedical Research.