Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension

作者：Silvia Faravelli, Matteo Campioni, Martina Palamini, Anselmo Canciani, Antonella Chiapparino, Federico Forneris · 发表于：BIO-PROTOCOL · 年份：2021 · DOI：10.21769/bioprotoc.3998 · 被引用次数：20 · 研究领域：Viral Infectious Diseases and Gene Expression in Insects、CRISPR and Genetic Engineering、CAR-T cell therapy research

Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In this regard, human embryonic kidney 293 (HEK293) cells constitute one of the main standard lab-scale mammalian hosts for recombinant protein production since these cells are relatively easy to handle, scale-up, and transfect. Here, we present a detailed protocol for the cost-effective, reproducible, and scalable implementation of HEK293 cell cultures in suspension (suitable for commercially available HEK293 cells, HEK293-F) for high-quantity recombinant production of secreted soluble multi-domain proteins. In addition, the protocol is optimized for a Monday-to-Friday maintenance schedule, thus simplifying and streamlining the work of operators responsible for cell culture maintenance.Graphic abstract:Schematic overview of the workflow described in this protocol, [摘要]重组蛋白是一个对于不同的应用范围从药物到临床过多必不可少里程碑，和哺乳动物细胞系是目前优选的系统，以获得大量的目标蛋白质中由于它们的高电平后-翻译后修饰一个nd可管理的大规模生产。在这方面，人胚胎肾293（HEK293）细胞构成的一个主标实验室规模的哺乳动物宿主小号用于重组蛋白质生产，因为这些细胞是相对容易处理，按比例放大，并且转染。在这里，我们提出了一个详细的协议用于具有成本效益的，可再现的，在悬浮液和可扩展执行的HEK293细胞培养物（适于市售的HEK293细胞，HEK293-F），用于高量重组生产分泌的可溶性多域蛋白。此外，该协议为星期一到星期五的维护计划进行了优化，从而简化和STREA毫升进不去负责细胞培养维护人员的工作。图形摘要：该协议中描述的工作流程的示意图 [背景]高品质的重组蛋白的大的量用于所必需的多种试剂的字段范围从体外生化研...