Cloning And Expression Of Mgg_00245 Gene Isolated From Magnaporthe Oryzae On Escherichia Coli Bl21 (De3)

Abstract

Magnaporthe oryzae is a fungus causing rice blast disease. According to a study by Soanet et al 2012, the mgg_00245 transcription was highly regulated during the maturation stage of the appressorium. After looking more closely at the amino acid sequence of mgg_00245, we suspected that the mgg_00245 gene fragment has potential to code for a Chitin-specific polysaccharide monooxygenase (PMO) enzyme and may be involve in the pathogenesis of fungus Magnaporthe oryzae. For more in-depth research on the nature, activity, and potential application of this new enzyme, the mgg_00245 gene was cloned and expressed to receive the target protein. In this study, mgg_00245 gene was expressed in E. coli BL21(DE3), using pET22b under the control of T7 promoter. The expression of mgg_00245 gene induced under 100μM IPTG within 20 hours, target protein was detected on SDS-PAGE gel at 27 kDa. To confirm the expression, the protein was purified on affinity chromatography and showed the His-Tag protein corresponding to 27 kDa. Protein concentration was determined by Bradford method. As a result, we had successfully cloned the E. coli BL21 (DE3) that contain gene encoding for mgg_00245. After DNA sequencing, the sequence of sample 6 and sample 11 are exactly 100% as mgg_00245 optimize sequence. Protein concentration of sample 6 and sample 11 were 146.28 ug/mL and 304.05 ug/mL, respectively. The results showed that the mgg_00245 gene from Magnaporthe oryzae was successfully expressed on Escherichia coli BL21(DE3).