gms | German Medical Science

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

08. - 11.05.2004, Lübeck

Evidence for protease activity of the SARS Unique Domain (SUD)

Poster

  • Stefanie Tech - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • presenting/speaker Christian L. Schmidt - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • Doris Mutschall - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • Silke Schmidtke - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • Ralf Moll - Institute of Biochemistry, University of Lübeck, Lübeck, Germany
  • John Ziebuhr - Institute of Virology and Immunology, University of Würzburg, Würzburg, Germany
  • Parvesh Wadhwani - Institute for Organic Chemistry, University Karlsruhe, Karlsruhe, Germany
  • Anne S. Ulrich - Institute for Organic Chemistry, University Karlsruhe, Karlsruhe, 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. Doc04sarsP11.02

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/sars2004/04sars130.shtml

Veröffentlicht: 26. Mai 2004

© 2004 Tech et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

The analysis of the genome of the SARS coronavirus revealed a unique sequence element, the so-called SARS unique domain (SUD, Snijder [1] ). Despite extensive database searches, no significant similarities, or conserved sequence motives were detectable. To elucidate the function of this part of the polyprotein, we constructed clones for the expression of residues 1166 to 1544 (full length version) and 1207 to 1544 (∆1-43 version), fused to N- or C- terminal histidine tags in E. coli. The recombinant proteins were purified by metal affinity- and ion exchange- chromatography.

Size exclusion chromatography revealed the presence of two oligomeric states of the full-length SUD protein: A form with a native molecular mass of 120 kD, representing a dimer or a trimer, and a form with a mass of 250 kD, potentially being a pentamer or hexamer.

Both forms of SUD are fairly unstable and subject to rapid proteolysis. The full-length form reproducibly formed detectable intermediates, hinting at a function as a processing protease. This putative autoproteolytic activity was influenced by several components as demonstrated in Figure 1 [Fig. 1] and showed a weak sensitivity to serine protease inhibitors such as Pefablock. Both forms of the recombinant SUD proteins showed anomalies in the UV / Vis spectrum suggesting the presence of a cofactor. In the case of the ∆1-43 protein, the apo- and holo-forms could be separated, enabling us to calculate the absorbance spectrum of the co-factor (figure 2 [Fig. 2]).

In summary, there is substantial evidence for a function of the SUD protein as a third, novel protease of the SARS coronavirus.


References

1.
Snijder et al. (2003) JMB, 331, 991 - 1004.