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  1. Article ; Online: Transient transfection and purification of SARS-CoV-2 spike protein from mammalian cells

    Victoria Stalls / Katarzyna Janowska / Priyamvada Acharya

    STAR Protocols, Vol 3, Iss 3, Pp 101603- (2022)

    2022  

    Abstract: Summary: 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. ... ...

    Abstract Summary: 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 Stalls et al. (2022), Gobeil et al. (2022), Edwards et al. (2021), and Henderson et al. (2020). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
    Keywords Biophysics ; Microbiology ; Protein Biochemistry ; Protein expression and purification ; Science (General) ; Q1-390
    Subject code 612
    Language English
    Publishing date 2022-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction

    Sophie M.-C. Gobeil / Katarzyna Janowska / Shana McDowell / Katayoun Mansouri / Robert Parks / Kartik Manne / Victoria Stalls / Megan F. Kopp / Rory Henderson / Robert J. Edwards / Barton F. Haynes / Priyamvada Acharya

    Cell Reports, Vol 34, Iss 2, Pp 108630- (2021)

    2021  

    Abstract: Summary: The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S ...

    Abstract Summary: The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic and are now the dominant form worldwide. Here, we explore S conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryoelectron microscopy (cryo-EM) structures reveal altered receptor binding domain (RBD) disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the RBDs in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, which harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 S conformational landscape and allostery and have implications for vaccine design.
    Keywords COVID-19 ; SARS-CoV-2 ; spike ; 2P ; D614G ; allostery ; Biology (General) ; QH301-705.5
    Subject code 612
    Language English
    Publishing date 2021-01-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

    Full text online

    More links

    Kategorien

    Inter-library loan at ZB MED

    Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

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