Molecular Cloning and Expression of Modified Allium Sativum Root Lectin in Pichia Pastoris X-33

Lectins or agglutinins are sugar-binding proteins that bind reversibly to specific mono- or oligo-saccharides, widely distributed in plants, animals, and microbes. The physiological role of lectins in plant growth and development and plant defense against pathogens, and insect pests. Plant lectins have a severe effect on the growth and development of insects. In this study, the Allium sativum root lectin (ASARI) gene was adopted from the National Centre for Biotechnology Informatics (Gene bank accession number AAB64238.1). The isolated ASARI gene was modified by deleting 84bp at 5’end and 126bp at the 3’ end of the gene to enhance the activity of the expressed protein. The modified mASARI gene was isolated by PCR, the 330bp gene was cloned into the pGEMT easy vector then cloned into the expression vector pGAPZαB and transformed into the host Pichia pastoris X33. The pGAPZαB vector contains an alpha-secretory signal that facilitates directly exporting the properly folded protein into the culture media. The modified recombinant garlic root lectin was successfully expressed in Pichia pastoris X33 and showed an expression of 12.1 kDa on SDS-PAGE and western blotting. It was purified by Ni+ NTA column chromatography in its native and functional characteristics. Additionally, the 12.1 kDa protein exhibited positive agglutination with rabbit erythrocytes at a concentration of 1.56 µg/ml.