Optimized Recombinant Expression and Purification of the SARS‐CoV‐2 Polymerase Complex Article Swipe
Ana Podadera
,
Lucas Campo
,
Fasih ur Rehman
,
Nikola Kolobaric
,
Adriana Zutic
,
Kenneth Ng
·
YOU?
·
· 2024
· Open Access
·
· DOI: https://doi.org/10.1002/cpz1.1007
YOU?
·
· 2024
· Open Access
·
· DOI: https://doi.org/10.1002/cpz1.1007
An optimized protocol has been developed to express and purify the core RNA‐dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2). The expression and purification of active core SARS‐CoV‐2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS‐CoV‐2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose‐binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS‐CoV‐2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Expression and production of SARS‐CoV‐2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol : Establishment and maintenance of insect cell cultures Basic Protocol 2 : Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3 : Purification of the SARS‐CoV‐2 core polymerase complex
Related Topics
Concepts
Recombinant DNA
Sf9
Biology
Polymerase
Fusion protein
RNA polymerase
Escherichia coli
Maltose-binding protein
T7 RNA polymerase
Computational biology
Cell biology
Biochemistry
Enzyme
Gene
Bacteriophage
Spodoptera
Metadata
- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.1002/cpz1.1007
- https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/cpz1.1007
- OA Status
- hybrid
- Cited By
- 1
- References
- 42
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4393045409
All OpenAlex metadata
Raw OpenAlex JSON
- OpenAlex ID
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https://openalex.org/W4393045409Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1002/cpz1.1007Digital Object Identifier
- Title
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Optimized Recombinant Expression and Purification of the SARS‐CoV‐2 Polymerase ComplexWork title
- Type
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articleOpenAlex work type
- Language
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enPrimary language
- Publication year
-
2024Year of publication
- Publication date
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2024-03-01Full publication date if available
- Authors
-
Ana Podadera, Lucas Campo, Fasih ur Rehman, Nikola Kolobaric, Adriana Zutic, Kenneth NgList of authors in order
- Landing page
-
https://doi.org/10.1002/cpz1.1007Publisher landing page
- PDF URL
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https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/cpz1.1007Direct link to full text PDF
- Open access
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YesWhether a free full text is available
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hybridOpen access status per OpenAlex
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https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/cpz1.1007Direct OA link when available
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Recombinant DNA, Sf9, Biology, Polymerase, Fusion protein, RNA polymerase, Escherichia coli, Maltose-binding protein, T7 RNA polymerase, Computational biology, Cell biology, Biochemistry, Enzyme, Gene, Bacteriophage, SpodopteraTop concepts (fields/topics) attached by OpenAlex
- Cited by
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1Total citation count in OpenAlex
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2025: 1Per-year citation counts (last 5 years)
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42Number of works referenced by this work
- Related works (count)
-
10Other works algorithmically related by OpenAlex
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