Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

Abstract

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Insights into the specificity for the interaction of the promiscuous SARS-CoV-2 nucleocapsid protein N-terminal domain with deoxyribonucleic acids

“…The gene sequences encoding the SARS-CoV-2 (NC_045512.2) nucleocapsid protein N-terminal domain (N-NTD; residues 44–180) and the N-NTD containing the C-terminal SR-rich motif (N-NTD-SR; residues 44–212) were codon-optimized, synthesized, and subcloned into pET28a by GenScript (Piscataway, USA). Expression and purification of these constructs were previously described by the international Covid19-NMR consortium ( https://covid19-nmr.de/ ) [33] . Briefly, pET28a-N-NTD and pET28a-N-NTD-SR were transformed into Escherichia coli BL21 (DE3).…”

Backbone and Ile, Leu, Val methyl group resonance assignment of CoV-Y domain of SARS-CoV-2 non-structural protein 3

“…We previously described expression and purification of nsp3 CoV-Y (residues 1638–1945) (Altincekic et al 2021 ). This construct provided good quality 1 H- 15 N TROSY-HSQC spectra but a long N-terminal unstructured region (approximately 30 residues) complicated the assignment.…”

“…The protein was purified as described previously (Altincekic et al 2021 ) with two modifications: (I) we added 1 mM of tris (2-carboxyethyl) phosphine (TCEP) after TEV-cleavage of the His 6 -tag to reduce cysteine side chains; (II) we changed the final buffer because the computed pI was reduced from 7.1 to 6.6 for the new shorter construct. All NMR samples were prepared in 20 mM MOPS buffer pH 6.4, 100 mM LiBr, 2 mM DTT and 0.01% NaN 3 .…”

Partial structure, dampened mobility, and modest impact of a His tag in the SARS-CoV-2 Nsp2 C-terminal region

“…To enable inhibitor screening and to uncover conformational preferences and dynamics, we have expressed and purified the 13 C, 15 N-labeled C-terminal region of Nsp2. The full production methodology of this and other SARS-CoV-2 proteins has been recently published (Altincekic et al 2021 ). Briefly, the DNA sequence coding for the segment K 557 EIIFLEGETLPTEVLTEEVVLK-TGDLQPLEQPTSEAVEAPLVGT 601 , from the C-terminus of Nsp2 was synthesized (GenScript, New Jersey, USA) and cloned in plasmid pET45b also coding for an N-terminal hexaHis tag and enterokinase cleavage site or in a pET28a-derived plasmid coding for an N-terminal thioredoxin domain, a hexaHis tag and a TEV-protease cleavage site.…”