Novel purification method and antibiotic activity of recombinant Momordica charantia MAP30

Ching-Dong Chang,Ping-Yuan Lin,Yo-Chia Chen,Wen-Ling Shih,Han-Hsiang Huang

doi:10.1007/s13205-016-0590-8

Abstract

Ribosome-inactivating proteins (RIPs) are a group of enzymes originally isolated from plants that possess the ability to damage ribosomes in an irreversible manner, leading to inhibition of protein synthesis in eukaryotic cells. In this study, we aimed to purify recombinant RIPs, investigate their function in the treatment of bacterial infection, and determine their toxicity in mice. We employed a pMAL protein fusion and purification system using E. coli transformed with a plasmid containing MBP-tagged MAP30 cDNA. MBP-tagged MAP30 was purified using a modified novel protocol to effectively produce highly active MAP30 of high purity. In an acute toxicity study in mice, no mortality occurred at doses lower than 1.25 mg/kg. MAP30 at both 0.42 and 0.14 mg/kg induced anti-MAP30 IgG, which reached a maximum titer at week 3. In conclusion, recombinant MAP30 prepared using our purification method possesses bioactivity, and has a synergistic bacteria-killing effect that can significantly reduce the required dosages of chloramphenicol and erythromycin. Therefore, when MAP30 is used in combination with chloramphenicol or erythromycin, it may of benefit in terms of reducing the side effects of the antibiotics, as lower concentrations of antibiotics are required.

Highlights

Ribosome-inactivating proteins (RIPs) are N-glycosidases that were initially isolated from plants
RIPs can be classified into three groups based on their primary structure: type 1 RIPs are single-chain proteins with a molecular weight of about 30 kD; type 2 RIPs are proteins of 60–65 kD that are composed of two chains, an A-chain with rRNA N-glycosidase activity linked to a lectin-like B chain via a disulfide bond; and type 3 RIPs are synthesized as proenzymes and require proteolysis to remove a short internal segment, transforming the inactive precursor into an active RIP (Xia et al 2003; Girbes et al 2004)
MAP30 purified from Mormodica charantia (MC) has been demonstrated to have a high activity against the herpes simplex virus (HSV) (Bourinbaiar and Lee-Huang 1996), and has been shown to inhibit the proliferation of AIDS-related lymphoma cells infected with herpes simplex virus-8 (HHV-8) by downregulating cellular gene expressions related to uncontrolled proliferation (Sun et al 2001)

Summary

Introduction

Ribosome-inactivating proteins (RIPs) are N-glycosidases that were initially isolated from plants. MAP30 purified from MC has been demonstrated to have a high activity against the herpes simplex virus (HSV) (Bourinbaiar and Lee-Huang 1996), and has been shown to inhibit the proliferation of AIDS-related lymphoma cells infected with herpes simplex virus-8 (HHV-8) by downregulating cellular gene expressions related to uncontrolled proliferation (Sun et al 2001). It inhibits the cell proliferation of a panel of tumor cells in vitro and in vivo (Fang and Ng 2011; Fang et al 2012a, 2012b). Previous research has indicated that anti-viral activities are the best-characterized effects of MAP30, especially against HIV

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Conclusion