Chang Ching-Dong, Lin Ping-Yuan, Chen Yo-Chia, Huang Han-Hsiang, Shih Wen-Ling
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Department of Biological Science and Technology, National Pingtung University of Science and Technology, 1, Shuefu Rd., Neipu, Pingtung, 91201, Taiwan.
3 Biotech. 2017 May;7(1):3. doi: 10.1007/s13205-016-0590-8. Epub 2017 Apr 7.
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.
核糖体失活蛋白（RIPs）是最初从植物中分离出来的一组酶，它们具有以不可逆方式损伤核糖体的能力，从而导致真核细胞中蛋白质合成受到抑制。在本研究中，我们旨在纯化重组RIPs，研究它们在治疗细菌感染中的功能，并确定它们对小鼠的毒性。我们采用了一种pMAL蛋白融合和纯化系统，使用用含有MBP标签的MAP30 cDNA的质粒转化的大肠杆菌。使用改良的新方案纯化MBP标签的MAP30，以有效地产生高纯度的高活性MAP30。在小鼠急性毒性研究中，剂量低于1.25mg/kg时未发生死亡。0.42mg/kg和0.14mg/kg的MAP30均诱导产生抗MAP30 IgG，在第3周达到最大滴度。总之，使用我们的纯化方法制备的重组MAP30具有生物活性，并且具有协同杀菌作用，可以显著降低氯霉素和红霉素的所需剂量。因此，当MAP30与氯霉素或红霉素联合使用时，可能有利于减少抗生素的副作用，因为所需的抗生素浓度较低。
