Transient transfection and purification of SARS-CoV-2 spike protein from mammalian cells.
Transient transfection and purification of SARS-CoV-2 spike protein from mammalian cells.
复制标题
DOI：
10.1016/j.xpro.2022.101603
复制
发表时间：
2022-09-16
期刊：
影响因子：
--
通讯作者：
Acharya, Priyamvada
中科院分区：
文献类型：
--
作者：
Stalls, Victoria;Janowska, Katarzyna;Acharya, Priyamvada
SARS-CoV-2 刺突 (S) 蛋白胞外域纯化可能具有挑战性，工程变异和自然变异通常会导致产量较低。在这里，我们提出了 SARS-CoV-2 S 胞外域的详细转染和纯化方案。我们描述了如何利用热变性时胞外域固有荧光的高度敏感和特征变化来追踪纯化过程中的蛋白质产量。此外，我们详细介绍了纯化的几个优化方面，包括时间和温度。该方案有利于 SARS-CoV-2 S 胞外域的一致、高质量制备。有关该协议的使用和执行的完整详细信息，请参阅和。制备高质量 SARS-CoV-2 刺突 (S) 蛋白胞外域样品 通过差示扫描荧光法 (DSF) 跟踪纯化过程中的 S 蛋白产量 冷敏感 S 蛋白的瞬时转染和纯化 出版商说明：进行任何实验方案都需要遵守当地实验室安全和道德机构指南。 SARS-CoV-2 刺突 (S) 蛋白胞外域纯化可能具有挑战性，工程变异和自然变异通常会导致产量较低。在这里，我们提出了 SARS-CoV-2 S 胞外域的详细转染和纯化方案。我们描述了如何利用热变性时胞外域固有荧光的高度敏感和特征变化来追踪纯化过程中的蛋白质产量。此外，我们详细介绍了纯化的几个优化方面，包括时间和温度。该方案有利于 SARS-CoV-2 S 胞外域的一致、高质量制备。
SARS-CoV-2 spike (S) protein ectodomain purification can be challenging, with engineered and natural variations often resulting in lower yields. Here, we present a detailed transfection and purification protocol for the SARS-CoV-2 S ectodomain. We describe how to trace protein yields during purification using highly sensitive and characteristic changes in S ectodomain intrinsic fluorescence upon thermal denaturation. Additionally, we detail several optimized aspects of the purification including timing and temperature. This protocol facilitates consistent, high-quality preparations of the SARS-CoV-2 S ectodomain. For complete details on the use and execution of this protocol, please refer to, and. Preparation of high-quality SARS-CoV-2 spike (S) protein ectodomain samples Track S protein yields during purification by differential scanning fluorimetry (DSF) Transient transfection and purification of the cold-sensitive S protein Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. SARS-CoV-2 spike (S) protein ectodomain purification can be challenging, with engineered and natural variations often resulting in lower yields. Here, we present a detailed transfection and purification protocol for the SARS-CoV-2 S ectodomain. We describe how to trace protein yields during purification using highly sensitive and characteristic changes in S ectodomain intrinsic fluorescence upon thermal denaturation. Additionally, we detail several optimized aspects of the purification including timing and temperature. This protocol facilitates consistent, high-quality preparations of the SARS-CoV-2 S ectodomain.
登录
查看更多内容
影响因子：
16
作者：
Gobeil SM;Henderson R;Stalls V;Janowska K;Huang X;May A;Speakman M;Beaudoin E;Manne K;Li D;Parks R;Barr M;Deyton M;Martin M;Mansouri K;Edwards RJ;Eaton A;Montefiori DC;Sempowski GD;Saunders KO;Wiehe K;Williams W;Korber B;Haynes BF;Acharya P
通讯作者：
Acharya P
影响因子：
8.8
作者：
Gobeil SM;Janowska K;McDowell S;Mansouri K;Parks R;Manne K;Stalls V;Kopp MF;Henderson R;Edwards RJ;Haynes BF;Acharya P
通讯作者：
Acharya P
影响因子：
16.8
作者：
Henderson, Rory;Edwards, Robert J.;Acharya, Priyamvada
通讯作者：
Acharya, Priyamvada
影响因子：
16.8
作者：
Edwards RJ;Mansouri K;Stalls V;Manne K;Watts B;Parks R;Janowska K;Gobeil SMC;Kopp M;Li D;Lu X;Mu Z;Deyton M;Oguin TH 3rd;Sprenz J;Williams W;Saunders KO;Montefiori D;Sempowski GD;Henderson R;Munir Alam S;Haynes BF;Acharya P
通讯作者：
Acharya P
影响因子：
56.9
作者：
Wrapp, Daniel;Wang, Nianshuang;McLellan, Jason S.
通讯作者：
McLellan, Jason S.