Molecular Cloning, Expression and On-Column Refolding Of Recombinant Modified Allium Sativum Root Lectin in E. coli/BL21

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/m

Molecular Cloning, Expression, and On-Column Refolding of Recombinant Allium sativum Root Lectin in E. coli /BL21

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 &nbsp

Correction to: Transient expression of SbDhr2 and MeHNL in Gossypium hirsutum for herbivore deterrence assay with Spodoptera litura

An amendment to this paper has been published and can be accessed via the original article.</jats:p

Functional characterization, homology modeling and docking studies of beta-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)

Glycosyl hydrolase family 1 beta-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving beta-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this beta-glucosidase were found to be 45 A degrees C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K (m) and V (max) were found to be 38.59 mu M and 0.8237 mu M/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 mu M and 0.1037 mu M/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (Delta G) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate