Automated overexpression and isotopic labelling of biologically active oncoproteins in the cyanobacterium Anabaena sp. PCC 7120. - Inserm - Institut national de la santé et de la recherche médicale Access content directly
Journal Articles Biotechnology and Applied Biochemistry Year : 2008

Automated overexpression and isotopic labelling of biologically active oncoproteins in the cyanobacterium Anabaena sp. PCC 7120.

Abstract

We have previously shown the cyanobacterium Anabaena sp. PCC 7120 to be a suitable host for the production of isotopically labelled recombinant proteins using the nitrate-inducible nir expression system. However, the expression of toxic proteins such as oncoproteins proved to be difficult, as expression levels decreased shortly after induction, while growth continued. To overcome this limitation, we have developed a method of auto-induction of the nir promoter in which cells are grown to high cell density in a bioreactor in the presence of ammonium and nitrate. Since ammonium is the preferred nitrogen source and acts as a repressor of the nir promoter, induction occurs only when the ammonium had been depleted. Using this novel auto-induction method, both oncoproteins E6 and gankyrin were expressed at high levels in a folded conformation and were shown to be biologically active after purification. Furthermore, under similar conditions of growth in auto-inducing medium, the use of (15)N- and (13)C-labelled mineral salts yielded isotopic enrichment of these proteins at levels above 95%, making them suitable for NMR-based structural analysis in a cost-effective manner.

Domains

Ethics

Dates and versions

inserm-00350862 , version 1 (07-01-2009)

Identifiers

Cite

Dominique Desplancq, Anne-Sophie Rinaldi, Helen Hörzer, Yu Ho, Hélène Nierengarten, et al.. Automated overexpression and isotopic labelling of biologically active oncoproteins in the cyanobacterium Anabaena sp. PCC 7120.. Biotechnology and Applied Biochemistry, 2008, 51 (Pt 1), pp.53-61. ⟨10.1042/BA20070276⟩. ⟨inserm-00350862⟩
169 View
0 Download

Altmetric

Share

Gmail Facebook X LinkedIn More