Identification and CRISPR/Cas9 Inactivation of the C1s Protease Responsible for Proteolysis of Recombinant Proteins Produced in CHO Cells.
Identification and CRISPR/Cas9 Inactivation of the C1s Protease Responsible for Proteolysis of Recombinant Proteins Produced in CHO Cells.
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负责 CHO 细胞中产生的重组蛋白水解的 C1s 蛋白酶的鉴定和 CRISPR/Cas9 灭活。
DOI：
10.1002/bit.27016
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发表时间：
2019
影响因子：
3.8
通讯作者：
Berman,PhillipW
中科院分区：
文献类型：
--
作者：
Li,SophiaW;Yu,Bin;Byrne,Gabriel;Wright,Meredith;O'Rourke,Sara;Mesa,Kathryn;Berman,PhillipW
中国仓鼠卵巢（CHO）细胞中重组蛋白表达相关的蛋白水解阻碍了包括HIV疫苗在内的生物制剂的开发。当在CHO细胞中表达时，重组HIV包膜蛋白gp120在中和抗体识别的关键表位上被丝氨酸蛋白酶进行蛋白水解剪切。对于来自B支病毒的包膜蛋白来说，问题尤其严重。B支病毒是在包括美国和欧洲在内的许多发达国家流行的主要遗传亚型。在本文中，我们已经确定了补体组分1 (C1s)，一种来自补体级联的丝氨酸蛋白酶，作为CHO细胞中负责gp120蛋白水解的蛋白酶。在CHO细胞系中，CRISPR/Cas9敲除C1s蛋白酶被证明可以消除对重组表达的gp120的蛋白水解活性。此外，C1s - / - MGAT1 - CHO细胞系敲除了C1s蛋白酶和MGAT1糖基转移酶，使B支分离物（BaL - rgp120）产生了未剪切的gp120，并在gp120上富集了甘露糖- 5聚糖，这是结合多种广泛中和的单克隆抗体（bN - mab）所必需的。这项技术的可用性将允许在世界上B支病毒流行的区域扩大和测试多种疫苗概念。此外，C1s蛋白酶引起的蛋白水解问题表明，在CHO细胞中，更需要C1s缺陷的CHO细胞系来表达其他易受丝氨酸蛋白酶活性影响的重组蛋白。同样，本文描述的在CHO细胞系中识别和敲除C1s的工作流程可以应用于补救其他CHO蛋白酶对生物制剂的蛋白水解。
Proteolysis associated with recombinant protein expression in Chinese Hamster Ovary (CHO) cells has hindered the development of biologics including HIV vaccines. When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1's (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s−/−MGAT1−CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL‐rgp120) and enriched for mannose‐5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN‐mAbs). The availability of this technology will allow for the scale‐up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation. Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s‐deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases.