Polymerase Chain Reaction (PCR) based cloning of Mitogen Activated Protein Kinase (MAPK3) in expression vector for isolation and purification of MAPK3 protein

Alternaria blight incited by a fungus Alternaria brassicae causes significant economic losses in Brassica crops depending upon the severity of the disease incidence. MAPK3 which is one of the important components of MAP Kinase signaling pathway is known to be involved in plant defense in Arabidopsis thaliana which has interstingly been shown as a host for Alternaria blight disease. Hence, this MAP kinase was used as target for raising antibodies against it for it"s immunological detection during progression of Alternaria blight disease.Full length cDNA clone for MAPK3, was amplified from Arabidopsis thaliana plants, eluted from gel and cloned into pGEX4T2 expression vector carrying the glutathione-S transferase (GST) tag. Now the recombinant pGEX4T2 plasmid was introduced into Bl21 strain of E. coli for efficient synthesis of MAP Kinase3 protein. Then, IPTG induced synthesis of GST-MAP Kinase fusion protein was monitored through analysis of total bacterial protein by SDS- PAGE. After purification, these polyclonal antibodies can be used to purify respective MAP Kinase 3 from Brassica and to study regulation of MAP Kinases at translational level.