Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

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Tekpınar A., Madru C., Rosario S., Czernecki D., Bruˆ Le S., Sauguet L., ...Daha Fazla

PLOS ONE, cilt.16, sa.4, ss.1-12, 2021 (SCI-Expanded)

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 16 Sayı: 4
  • Basım Tarihi: 2021
  • Doi Numarası: 10.1371/journal.pone.0250610
  • Dergi Adı: PLOS ONE
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Agricultural & Environmental Science Database, Animal Behavior Abstracts, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, Biotechnology Research Abstracts, Chemical Abstracts Core, EMBASE, Food Science & Technology Abstracts, Index Islamicus, Linguistic Bibliography, MEDLINE, Pollution Abstracts, Psycinfo, zbMATH, Directory of Open Access Journals
  • Sayfa Sayıları: ss.1-12
  • Van Yüzüncü Yıl Üniversitesi Adresli: Evet

Özet

To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus

2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules

are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent

RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a

multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that

ensure the ~30 kb RNA genome’s transcription and replication. The main strategies

employed so far for the overproduction of RdRp consist of expressing and purifying the

three subunits separately before assembling the complex in vitro. However, nsp12 shows

limited solubility in bacterial expression systems and is often produced in insect cells. Here,

we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli,

using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp

shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA

polymerization activity was measured using primer-extension assays showing that the purified

enzyme is functional. The purification protocol can be achieved in one single day, surpassing

in speed all other published protocols. Our construct is ideally suited for screening

RdRp and its variants against very large chemical compounds libraries and has been made

available to the scientific community through the Addgene plasmid depository (Addgene ID:

165451).