Expression and purification of a novel kazal-type protease inhibitor of Arabidopsis thaliana

PARIANI, S.; ACOSTA, D.; DUSCHAK V G; BUSI, MV.; GOMEZ-CASATI, D.; CLEMENTE, M.

Rosario

Congreso; Reunion SAIB 2014; 2014

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Serine protease inhibitors are widely distributed in plant kingdom. The physiological role of these proteins includes regulation of endogenous proteinases, mobilization of storage proteins and protection against several pathogens. To date there are no reported kazal serine protease inhibitors in plants. However, an in silico analysis revealed the presence of two open reading frames which encode for two kazal-like proteins in Arabidopsis thaliana. The aim of the present work was the expression and purification of a putative kazal serine protease inhibitor, which was named A. thaliana Kazal-type protease Inhibitor 1 (AtKPI-1). We determined the appropriated host for an efficient expression of the rAtKPI-1 in bacteria testing E. coli BL21 pLys, BL21 (DE3) and C41 (DE3) strains. After addition of IPTG, a protein with apparent molecular mass of 12 kDa was detected in BL21 (DE3), while no significant expression levels of rAtKPI-1 was found in BL21 pLys and C41 strains. The presence of the rAtKPI-1T eluted from Ni-NTA chromatography column was confirmed by SDS?PAGE and western blot analysis. From 1 L LB of bacterial culture corresponding to ~ 8 g of E. coli cells, 22 mg of purified rAtKPI-1T was obtained. We report an efficient method for the production of large amount of highly purified AtKPI-1T required for studies concerning the biological functions of the members of the kazal family.