Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays
Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays
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DOI：
10.1016/j.pep.2020.105686
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发表时间：
2020-10-01
影响因子：
1.6
通讯作者：
Gillette, William
中科院分区：
文献类型：
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作者：
Esposito, Dominic;Mehalko, Jennifer;Gillette, William
SARS-CoV-2 刺突三聚体是多种血清学检测的主要抗原，对于确定人群中 SARS-CoV-2 的暴露程度至关重要。在开发出稳定的细胞系以提高哺乳动物细胞培养物中这种分泌蛋白的效价之前，HEK293 细胞瞬时转染产生的刺突蛋白的低产量将是这些测定的限制因素。为了提高刺突蛋白的产量并支持血清学测定中对抗原的高需求，我们通过改变孵育温度、收获时间、层析策略和最终蛋白质操作来研究几种重组蛋白表达变量。通过这项研究，我们开发了一种简化而强大的纯化策略，对于两种常用形式的 SARS-CoV-2 刺突蛋白，每升表达培养物始终能产生 5 毫克蛋白质。我们证明这些蛋白质形成表现良好的稳定三聚体，并且在多个蛋白质生产批次的血清学测定中始终发挥作用。
The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.