Gene cloning and expression of map30 from Mara Khee Nok Monordica charantia Linn. in Escherichia coli

Abstract

MAP30 is a 30 kDa protein that was identified and purified from bitter melon (Momordica charantia). MAP30 contains protein synthesis inhibition activity and topoisomerase-like activity, these activities can inhibit HIV virus by capable of acting multiple stages of the vial life cycle. In Thailand, Mara Khee Nok (Thai bitter melon) is wildly distributed and consumed as food. To clone map30gene from this local variety, map30gene was first amplified from Mara Khee Nok genomic DNA by Polymerase Chain Reaction using specific primers designed from known map30sequence. The putative gene was cloned into pUC18 and electrotransformed to E.coliJM109. The resulting, the product about 800 bp correlated with mature map30in size. It was sequenced and the amino acids were deduced. The sequences were vary similar (2-8 nucleotides and 1-3 amino acids difference) to those reported in the DNA databank. The result implied that MAP30 is a highly conserved protein. Furthersubcloned to pET19b vector for high expression in E.coli-Rosetta DE3 (pLysS), the recombinant MAP30 was found as soluble and inclusion bodies forms. The inclusion bodies was solubilized with 8 M urea, refolded and purified by CM-Sephadex C50 and followed with Phosphocellulose P11. A purified protein of 30 kDa was obtained but it did not contain the topoisomerase activity. Purification of soluble MAP30 showed that most of MAP30 could be precipitated with 30 60% saturated ammonium sulfate. Purification by CM-Sephadex C-50 column gave two unbounded protein peaks, CmI and CmII. Topoisomerase activity was found in CmII fraction. The protein was purified to homogeneity from Phosphocellulose P11 column as confirm by SDS-PAGE analysis. The purified recombinant protein was confirmed to be MAP30 by N-terminal amino acids sequencing of the first 20 residues. The estimated molecular weight from computation program was 29.62 kDa. The minimal protein concentration that could show topoisomerase activity was 0.85 micrometre.