Sequencing and in silico analysis of the curcin isoforms in Jatropha (Jatropha curcas L.), and cloning in the expression vector

Abstract

Nowadays Jatropha(Jatropha curcas L.) is gaining world-wide importance due to its seed oil which is becoming a promising source of biodiesel. It can be grown on degraded and marginal land areas. It is also suitable in different types of soil conditions, rainfall and climates. It contains number of significant commercial metabolites which could be used in making cosmetics, soaps, fertilizers and lubricants. Presence of toxic substances in seeds like curcin and phorbol esters, limit their applications. Curcin, a major toxin protein, is present in seeds of Jatropha and other tissues. This is primarily a ribosome inactivating proteins (RIPs) which produces cytotoxic effects. Significantly, curcin protein has some pharmacological importance i.e. since its protein can be used as anti-tumor, anti-HIV, anti-viral and immune-suppressive agents. The present study focused on a curcin cDNA clone in pMD 20-Tvector namely Curcin 2A-17 having 99% sequence identity with a curcin isoform i.e Curcin2A (GenBank protein id: ADN39428). Various facile bioinformatics tools were used to study different attributes generating 3-D models, protein motifs, phylogenetic tree, Ramchandran plot, Hydropathy characters of the curcin isoform encoded by Curcin 2A-17, apart from studying its relatedness and divergence from other curcin isoforms. In order to clone the above Curcin cDNA in a suitable expression vector, the following efforts were made: Good quality of plasmid DNA i.e., protein expression vector, pET28a DNA was isolated. Curcin cDNA clone, approx. ~1.0 kb BamHI-HindIII fragment was eluted by gel extraction technique. Then it was cloned in to pET28avector digested with BamHI-HindIII. A few putative recombinant clones were isolated which will be duly characterized and used for recombinant protein expression.