High yields of stable and highly pure nucleocapsid proteins of different hantaviruses can be generated in the yeast Saccharomyces cerevisiae

Institute of Biotechnology, V. Graiciuno 8, LT-2028 Vilnius, Lithuania.
Journal of Biotechnology (Impact Factor: 2.88). 09/2004; 111(3):319-33. DOI: 10.1016/j.jbiotec.2004.04.010
Source: PubMed

ABSTRACT Recently, the high-level expression of authentic and hexahistidine (His)-tagged Puumala (strain Vranica/Hällnäs) hantavirus nucleocapsid protein derivatives in the yeast Saccharomyces cerevisiae has been reported [Dargeviciute et al., Vaccine, 20 (2002) 3523-3531]. Here we describe the expression of His-tagged nucleocapsid proteins of other Puumala virus strains (Sotkamo, Kazan) as well as Dobrava (strains Slovenia and Slovakia) and Hantaan (strain Fojnica) hantaviruses using the same system. All nucleocapsid proteins were expressed in the yeast S. cerevisiae at high levels. The nucleocapsid proteins can be easily purified by nickel chelate chromatography; the yield for all nucleocapsid proteins ranged from 0.5 to 1.5 mg per g wet weight of yeast cells. In general, long-term storage of all nucleocapsid proteins without degradation can be obtained by storage in PBS at -20 degrees C or lyophilization. The nucleocapsid protein of Puumala virus (strain Vranica/Hällnäs) was demonstrated to contain only traces of less than 10 pg nucleic acid contamination per 100 microg of protein. The yeast-expressed nucleocapsid proteins of Hantaan, Puumala and Dobrava viruses described here represent useful tools for serological hantavirus diagnostics and for vaccine development.

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Available from: Jonas Schmidt-Chanasit, Aug 25, 2015
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    • "The gene fragments encoding different polypeptides of hMPV N were subcloned into pFX7-6His (Razanskiene et al., 2004) by using plasmid hMPV N pFX7 as the template for the PCRs, resulting in plasmids pFX7-6His-hMPV N (90–395), pFX7-6His-hMPV N (1–365), pFX7-6His-hMPV N (1–335), pFX7-6His-hMPV N (1–305), pFX7-6His-hMPV N (1–275) and pFX7-6His-hMPV N (1–245). The primer pairs used were: 90-F and 395-R to amplify the hMPV N (90–395) construct, 1-F and 365-R to amplify the hMPV N (1–365) construct, 1-F and 335-R to amplify the hMPV N (1–335) construct, 1-F and 305-R to amplify the hMPV N (1–305) construct, 1-F and 275-R to amplify the hMPV N (1–275) construct, and 1-F and 245-R to amplify the hMPV N (1–245) construct. "
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    • "Immunohistochemistry using serum 371 against TioV N protein (A) and 372 against Menangle virus N protein (B), counterstained with haematoxylin. Scale-bar 50 ␮m. of other viruses generated in yeast expression systems (Sasnauskas et al., 1999; Samuel et al., 2002; Razanskiene et al., 2004; Juozapaitis et al., 2005, 2007a, 2007b, 2008). "
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