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Cloning, expression and purification of resistance gene analogue RGPM 301 from pearl millet in Escherichia coli

Mariswamy Veena, Prasad Melvin, Sekhar Shailasree, Kukkundoor Ramachandra Kini.

Abstract
Plants combat their pathogens with an array of defense responses. One of the key mechanisms involves products of resistance (R) genes which are responsible for recognition of effector molecules from pathogens and subsequent triggering of defense responses. Resistance gene analogues (RGAs) containing the specific conserved domains of R-genes are isolated from various plants using degenerate oligonucleotide primer based PCR approach. In an earlier study, RGPM 301, an RGA from pearl millet shown to be involved in resistance mechanism against downy mildew disease was isolated and characterized. In the present study, RGPM 301 containing an open reading frame (ORF) of 992 amino acids was cloned into pRSET A expression vector and expressed in Escherichia coli as a Hig-tag fusion protein. The recombinant RGA RGPM 301 was purified to near homogeneity using the Nickel-CL agarose column. Its molecular mass was found to be 120 kDa when separated on the SDS-PAGE which was confirmed by western blotting analysis using the anti-His antibody. The purified protein was subjected to in-gel trypsin digestion followed by mass spectrometric analysis for the confirmation of its identity. These findings facilitate further studies on the exact role of this RGA in the pearl millet downy mildew host pathogen system.

Key words: : pRSET A, BL21 (DE3), IPTG, CC-NBS-LRR, Affinity purification



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