Bone and Joint Research Unit, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom.
J Biotechnol. 2012 Oct 31;161(3):269-77. doi: 10.1016/j.jbiotec.2012.07.003. Epub 2012 Jul 23.
The development of novel protein therapeutics relies on the ability to express appreciable amounts of correctly folded recombinant proteins. Latent IFN-β is engineered using the latency-associated peptide (LAP) of transforming growth factor β1 (TGF-β1) to maintain IFN-β in a biologically inactive form until such time as it is released at sites of inflammation by matrix metalloproteinase activity (see Adams et al., 2003). CHO cells cultured in suspension were used for expression of latent IFN-β to allow medium scale transient transfection. However, the recombinant protein expressed in this system consisted of a mixture of properly linked disulphide dimers and monomers. The ratio of dimer:monomer produced could be significantly altered towards increased dimer production by the addition of L-cystine to the CHO culture medium. The total yield of latent IFN-β was increased by co-transfection of plasmid coding for the simian virus (SV) 40 large T antigen to the plasmid with the SV40 origin of replication expressing latent IFN-β DNA. These results provide valuable new insights for developing protocols to produce substantial quantities of latent cytokine dimers in CHO cells in suspension.
新型蛋白治疗药物的开发依赖于能够表达大量正确折叠的重组蛋白的能力。潜伏型 IFN-β 使用转化生长因子β1（TGF-β1）的潜伏相关肽（LAP）进行工程设计，以使 IFN-β保持生物活性，直到基质金属蛋白酶活性在炎症部位释放它（见 Adams 等人，2003）。CHO 细胞在悬浮培养中用于表达潜伏型 IFN-β，以允许中等规模的瞬时转染。然而，该系统中表达的重组蛋白由正确连接的二硫键二聚体和单体的混合物组成。通过向 CHO 培养基中添加 L-胱氨酸，可以显著改变二聚体：单体的产生比例，从而增加二聚体的产生。通过共转染编码猴病毒（SV）40 大 T 抗原的质粒，与表达潜伏型 IFN-β DNA 的 SV40 复制起点的质粒共转染，增加了潜伏型 IFN-β 的总产量。这些结果为开发在悬浮 CHO 细胞中大量生产潜伏细胞因子二聚体的方案提供了有价值的新见解。
