Haloalkaliphilic protease from an archaeon: overexpression in E. coli and Hfx. volcanii.

RUIZ DM; MAUPIN-FURLOW JA; DE CASTRO RE

Mar del Plata (Buenos Aires)

Congreso; XLIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).; 2007

Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular (SAIB)

The haloalkaliphilic archaeon Natrialba magadii (optimum growth in 20% NaCl, pH 12) secretes an organic solvent-tolerant protease denoted as NEP (Natrialba magadii Extracelular Protease) that has been biochemically characterized and the corresponding gene has been cloned and sequenced. The aim of this study was to overexpress the gene encoding NEP, nep, in two systems: Escherichia coli (bacteria) and Haloferax volcanii (haloarchaea). The coding region of nep was amplified by PCR from a genomic clone of Nab. magadii and the PCR product was cloned into pET24b expression vector generating  pET-nep and pET-nep-His6  constructs, which were expressed in E. coli BL21 (DE3) Rosetta cells. nep and nep-His6 were subcloned into a shuttle vector and transformed into Hfx. volcanii. NEP was expressed as an active enzyme in both systems, and the highest levels of extracellular protease activity were attained in Hfx. volcanii. Protease activity was not detected in the culture medium of E. coli cells suggesting that it was not secreted/processes. The recombinant enzyme produced in Hfx. volcanii, displayed similar biochemical properties and solvent-tolerance compared to the native enzyme. This research will contribute to optimize the high production of this extremozyme for basic studies and potential biotechnological applications. This work was supported by grants from ANPCyT, CONICET and UNMDP.