High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
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DOI：
10.3791/53568
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发表时间：
2015-12-01
影响因子：
1.2
通讯作者：
Barb, Adam
中科院分区：
文献类型：
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作者：
Subedi, Ganesh P.;Johnson, Roy W.;Barb, Adam
生产具有复杂翻译后修饰的重组蛋白的技术代表了结构和功能研究的主要挑战。瞬时转染的哺乳动物细胞系的快速建立和高回收率解决了这一障碍，并且是表达天然通过ER和高尔基体介导的分泌途径的蛋白质的有效手段。这是一种使用人HEK 293 F和HEK 293 S细胞系进行蛋白质表达的方案，所述细胞系用设计用于高蛋白质产量的哺乳动物表达载体转染。使用三种代表性糖蛋白证明了该系统的适用性，所述糖蛋白以每升培养物回收的95-120 mg纯化蛋白的产率表达。这些蛋白质是人Fc γ RIIIa和大鼠α 2-6唾液酸转移酶ST 6 GalI，两者均以N-末端GFP融合物表达，以及未修饰的人免疫球蛋白G1 Fc。这种强大的系统利用无血清培养基，适用于表达同位素富集的蛋白质和碳水化合物，用于使用质谱法和核磁共振光谱法进行结构研究。此外，可以通过添加小分子来调节N-聚糖的组成，以不降低产率的方式防止某些聚糖修饰。
The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc gamma RIIIa and the rat alpha 2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.