gms | German Medical Science

International Conference on SARS - one year after the (first) outbreak

08. - 11.05.2004, Lübeck

Expression of the N-terminal protein domain of SARS coronavirus RNA-dependent RNA polymerase (RdRp)


  • presenting/speaker Ralf Moll - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • Silke Schmidtke - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • Gerrit Volkmann - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • Franca Fuchs - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • Rolf Hilgenfeld - Institute of Biochemistry, University of Lübeck, Lübeck, Germany

International Conference on SARS - one year after the (first) outbreak. Lübeck, 08.-11.05.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04sarsP11.01

The electronic version of this article is the complete one and can be found online at:

Published: May 26, 2004

© 2004 Moll et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


A key player in replication and transcription of the single-stranded 30 kb genomic RNA of the SARS coronavirus is the RNA-dependent RNA polymerase (RdRp). The mature protein results from processing of the polyprotein pp1ab by the action of the viral main protease Mpro in the host cell ([1], [2]). As can be deduced from the conserved cleavage sites used by Mpro, the SARS-CoV RdRp comprises residues 4370 to 5301 of the polyprotein, pp1ab. It features a calculated molecular mass of 106 kD and a theoretical isoelectric point of 6.0. At least two regions within the whole RdRp sequence can be assigned putative protein domains by means of the localization of 7 canonical sequence motifs. Motifs A to G - assumed to line the active site of the polymerase - are embedded within the C-terminal part of the full-length protein and are part of the palm and finger subdomains. From a bioinformatic threading approach based on positions of the canonical sequences and known 3D structures of unrelated RdRps, a structural model has been constructed for residues 376 to 932 of the mature polymerase ([3]).

Most remarkable, however, is the presence of about 380 residues encompassing the N-terminal part of the SARS-CoV RdRp (RdRp-N). Strikingly, this part is unique for coronaviral RdRps, but is of yet unknown function due to the absence of characteristic sequence motifs. A functional relation to the C-terminal polymerase region or, more probably, interaction with other non-structural proteins of the coronaviral replicase complex is likely. In order to investigate the N domain of RdRp in more detail, it was heterologously expressed in different E. coli BL21(DE3) strains. Owing to an N-terminal histidine-tag sequence, RdRp-N could be purified as soluble protein on nickel-containing affinity resins. It shows a molecular mass of 33 kD in SDS-PAGE and was immuno-detected using anti-histidine antibodies. Protein patterns of gelfiltration experiments are of complex nature due to different oligomerization states, while the monomer is a minor form in the chromatograms. RdRp-N is a strongly acidic protein (pI 5.1) with low solubility between pH 4 and 7, but the domain exhibits higher solubility at pH 8. Buffer pH seems to be a more critical parameter for solubility than is ionic strength. SARS-CoV RdRp-N is easily proteolyzed by trypsin and chymotrypsin at pH 8, suggesting accessible cleavage sites on the protein surface, which are not hidden by oligomerization.

The expression protocol developed in this study allows functional in-vitro characterization, crystallization and X-ray structure determination of the RdRp-N, and, ultimately, structure-based drug discovery.


The authors are grateful to John Ziebuhr for providing the plasmid pETSCoV-Pol


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