Molecular Cloning, Expression, and On-Column Refolding of Recombinant Allium sativum Root Lectin in E. coli /BL21
DOI:
https://doi.org/10.53555/jaz.v43i1.5084Keywords:
Recombinant ASARI Lectin, PCR (Polymerase Chain Reaction), PET 30b (+), E. coli, SDS PAGE, refolding Western blotting, Ni-NTA column Chromatography, Agglutination, etc.Abstract
Lectins or agglutinins are the sugar-binding proteins that bind reversibly to specific mono- or oligo-saccharides. They are widely distributed in plants, animals, and microbes. The physiological role of lectins in plant growth and development, plant defense against pathogens, and insect pests. Plant lectins have a severe effect on the growth and development of insects. In this study, the gene of Allium sativum root lectin (ASARI) was adopted from the National Centre for Biotechnology Information. (Gene bank accession number AAB64238.1). The ASARI gene was amplified by PCR and cloned into the pET 30 b (+) vector with a C terminal His6 tag to get the over-expressed ASARI lectin. The His6 was used for the purification of Ni-NTA column chromatography. The over-expressed recombinant ASARI protein in E. coli /BL21 was an inactive inclusion body. The inclusion body contains lots of host cell proteins and cell components. To accomplish the active and native form of the protein from the inclusion body, it needs to be washed with different buffers to reduce the host cell components. For active and refolded forms of protein, it should be solubilized under denaturing conditions by (8M) urea. Then the protein was immobilized by metal affinity chromatography (IMAC) and gradually refolded by using a linear gradient of urea from 8.0 M to 0.0 M which showed that the protein was properly refolded. The 19.3 kDa protein showed positive agglutination with rabbit erythrocytes at a concentration of 12.5µg mg/ml
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