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Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding

The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic target...

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Autores principales: Struble, Lucas R., Smith, Audrey L., Lutz, William E., Grubbs, Gabrielle, Sagar, Satish, Bayles, Kenneth W., Radhakrishnan, Prakash, Khurana, Surender, El‐Gamal, Dalia, Borgstahl, Gloria E. O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Inc. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8996471/
https://www.ncbi.nlm.nih.gov/pubmed/35481636
http://dx.doi.org/10.1002/pro.4300
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author Struble, Lucas R.
Smith, Audrey L.
Lutz, William E.
Grubbs, Gabrielle
Sagar, Satish
Bayles, Kenneth W.
Radhakrishnan, Prakash
Khurana, Surender
El‐Gamal, Dalia
Borgstahl, Gloria E. O.
author_facet Struble, Lucas R.
Smith, Audrey L.
Lutz, William E.
Grubbs, Gabrielle
Sagar, Satish
Bayles, Kenneth W.
Radhakrishnan, Prakash
Khurana, Surender
El‐Gamal, Dalia
Borgstahl, Gloria E. O.
author_sort Struble, Lucas R.
collection PubMed
description The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).
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spelling pubmed-89964712022-04-15 Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding Struble, Lucas R. Smith, Audrey L. Lutz, William E. Grubbs, Gabrielle Sagar, Satish Bayles, Kenneth W. Radhakrishnan, Prakash Khurana, Surender El‐Gamal, Dalia Borgstahl, Gloria E. O. Protein Sci Methods and Applications The COVID‐19 pandemic caused by SARS‐CoV‐2 infection has led to socio‐economic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID‐19. SARS‐CoV‐2 spike is a target of interest for the development of therapeutic targets. We developed a robust SARS‐CoV‐2 S spike expression and purification protocol from insect cells and studied four recombinant SARS‐CoV‐2 spike protein constructs based on the original SARS‐CoV‐2 sequence using a baculovirus expression system: a spike protein receptor‐binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S‐RBD‐eGFP), spike ectodomain coupled to a fluorescent tag (S‐Ecto‐eGFP), spike ectodomain with six proline mutations and a foldon domain (S‐Ecto‐HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S‐Ecto‐HexaPro(‐F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S‐Ecto‐eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation). John Wiley & Sons, Inc. 2022-04-11 2022-05 /pmc/articles/PMC8996471/ /pubmed/35481636 http://dx.doi.org/10.1002/pro.4300 Text en © 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Methods and Applications
Struble, Lucas R.
Smith, Audrey L.
Lutz, William E.
Grubbs, Gabrielle
Sagar, Satish
Bayles, Kenneth W.
Radhakrishnan, Prakash
Khurana, Surender
El‐Gamal, Dalia
Borgstahl, Gloria E. O.
Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding
title Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding
title_full Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding
title_fullStr Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding
title_full_unstemmed Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding
title_short Insect cell expression and purification of recombinant SARS‐COV‐2 spike proteins that demonstrate ACE2 binding
title_sort insect cell expression and purification of recombinant sars‐cov‐2 spike proteins that demonstrate ace2 binding
topic Methods and Applications
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8996471/
https://www.ncbi.nlm.nih.gov/pubmed/35481636
http://dx.doi.org/10.1002/pro.4300
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