Molecular Cloning, Expression and On-Column Refolding Of Recombinant Modified Allium Sativum Root Lectin in E. coli/BL21
DOI:
https://doi.org/10.53555/jaz.v43i1.5086Keywords:
Recombinant ASARI Lectin, mASARI, PCR (Polymerase Chain Reaction), PET 30b (+), E. coli, SDS PAGE, refolding Western blotting, Ni-NTA column Chromatography, agglutination etc.Abstract
Lectins or agglutinins are 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 defence 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 modified (mASARI) by truncating 84bp at the 5’ end and 126bp at the 3’ end of the gene to enhance the binding activity to the target protein. The modified lectin was amplified by PCR and cloned into the pET 30 b (+) vector with C terminal His6 tag to get the over expressed modified mASARI lectin. The His6 is used for the purification of Ni-NTA column chromatography. But, the over expressed recombinant modified mASARI protein in E. coli is an inactive inclusion body. The inclusion body contains lots of host cell proteins and cell components. For receiving the active and native form of the protein the inclusion body has to be washed to reduce the host cell components. To get an active and refolded form of protein it should be solubilized under denaturing condition (8M) urea. Then the protein is immobilized by metal affinity chromatography (IMAC) and gradually refolded by using a linear gradient of urea from 8.0 M to 0.0 M showing that at least part of the protein was properly refolded. The 13.6 kDa protein showed positive agglutination with rabbit erythrocytes at a concentration of 6.25µg/ml
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Copyright (c) 2022 Satish Adat, Mayavati Patil , Shuban Rawal
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