Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells.
Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells.
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DOI：
10.1016/j.jbiotec.2020.12.005
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发表时间：
2021-01-20
影响因子：
4.1
通讯作者：
Durocher Y
中科院分区：
文献类型：
--
作者：
Stuible M;Gervais C;Lord-Dufour S;Perret S;L'Abbé D;Schrag J;St-Laurent G;Durocher Y
CHO细胞比293细胞更有效地表达三聚体SARS-CoV-2刺突蛋白。三聚化结构域和消除S1/S2弗林蛋白酶位点是获得刺突三聚体所必需的。转染后7天，在CHO细胞中获得前所未有的150 mg/L三聚体尖峰表达水平。SARS-CoV-2和相关病毒刺突蛋白的重组形式已被证明难以在哺乳动物细胞中以良好的产量生产。考虑到需要纯化刺突蛋白的潜在COVID-19诊断工具和治疗候选物的种类及其对正在进行的SARS-CoV-2研究的重要性，我们探索了刺突蛋白生产和纯化的新方法。比较了基于PEI介导转染CHO或HEK 293细胞的三种瞬时基因表达方法，以快速生产全长SARS-CoV-2刺突胞外域。使用DXB 11衍生的CHOBRI/55 E1细胞的高细胞密度方案比其他方法产生了显著更好的产量。表达了不同形式的刺突胞外域，包括野生型SARS-CoV-2序列和突变形式（以有利于表达稳定在融合前构象的全长刺突胞外域），有和没有融合到推定的三聚化结构域。还开发了一种有效的两步亲和纯化方法。最终，我们已经能够生产高度均匀的全长刺突的制备物，包括单体和三聚体，在收获的培养基中产量为100 - 150 mg/L。该方法的速度和生产率支持进一步开发用于重组刺突蛋白生产的基于CHO的方法。
CHO cells are more efficient than 293 cells for expressing trimeric SARS-CoV-2 spike protein. A trimerization domain and elimination of the S1/S2 furin site are essential to obtain spike trimers. Unprecedented trimeric spike expression levels of 150 mg/L are obtained in CHO cells 7 days post-transfection. Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100−150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.
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影响因子：
5.8
作者：
Qiu M;Shi Y;Guo Z;Chen Z;He R;Chen R;Zhou D;Dai E;Wang X;Si B;Song Y;Li J;Yang L;Wang J;Wang H;Pang X;Zhai J;Du Z;Liu Y;Zhang Y;Li L;Wang J;Sun B;Yang R
通讯作者：
Yang R
DOI：
10.1007/978-1-4939-8730-6_1
发表时间：
2018-01-01
期刊：
RECOMBINANT PROTEIN EXPRESSION IN MAMMALIAN CELLS: METHODS AND PROTOCOLS
影响因子：
--
作者：
L'Abbe, Denis;Bisson, Louis;Durocher, Yves
通讯作者：
Durocher, Yves
影响因子：
56.9
作者：
Patel, SD;Rajala, MW;Shapiro, L
通讯作者：
Shapiro, L
影响因子：
16.8
作者：
Tortorici, M. Alejandra;Walls, Alexandra C.;Veesler, David
通讯作者：
Veesler, David
影响因子：
2.2
作者：
Du, Lanying;Zhao, Guangyu;Jiang, Shibo
通讯作者：
Jiang, Shibo