Screening Peptide Inhibitors With The Isocitrate Lyase As The Target

Tuberculosis is a chronic communicable disease caused by Mycobacterium tuberculosis and spread around the world, about 200 million people died of TB each year all over the word. Previous studies showed that after taken up by macrophages, Mycobacterium tuberculosis becomes persistent in them and sustains life through the glyoxylate bypass.As isocitrate lyase (ICL) is a key rate-limiting enzyme in the the glyoxylate bypass and a decisive factor in mycobacterial persistence, it was selected as the target of new anti-tubercular drugs.In this study,the ICL gene of Mycobacterium tuberculosis H37RV was successfully cloned and expressed. The optimum conditions for ICL expression in E.coli BL21(DE3) was determined to be 0.25mmol/L IPTG induction for 4 h at 20℃. The expressed ICL was further purified on a Ni-NTA resin affinity chromatography. The target protein was eluted with 300mmol/L imidazole buffers.The recombinant ICL was purified in a highly active state with a specific activity of 24μmol·mg-1·min-1.The recombined ICL protein was used as the target, and the phage 7 peptide library was used to screen the phage which could be specifically combined with ICL. There were 4 rounds of screening.Based on the sequence comparison and ELISA results, three 7-peptide chains which had high affinity to ICL were selected. They are No.106 RHNPHQL, No.118 IISGKDS and No.331 RAQFIWP,respectively.The three 7-peptide chains were synthesized and put into reaction with ICL. All three peptides have inhibitory effects on the activity of ICL to different extent. The test showed that peptide 118 had strongest effects with the inhibitory rate of 47.94%, peptide 106 ranking the second with that of 44.57% and the last peptide 331 with that of 38.82%.This study provided lead compounds for the research and development of new peptide anti-TB drugs and provided the basis for the study and the development of the novel drugs against Mycobacterium tuberculosis.