Research on purification and activity characterization of AmPR-10 ribonuclease from Astragalus membranaceus / 中草药

ABSTRACT

Objective To establish a stable and rapid separation and purification method for Astragalus membranaceus (Am) pathogenesis-related protein-10 (AmPR-10) using an automatic intelligent protein purification system AKTA Avant 25, and analyze its physiochemical and biological activity. Methods Am was extracted by Tris-HCl buffer. The crude extract was captured by anion exchange chromatography, and finely separated by hydrophobic chromatography and gel filtration chromatography. The relative molecular weight of AmPR-10 was measured by MALDI-TOF/TOF mass spectrometry, the protein identification was determined by mass spectrometry and MS/MS Ion Search, the glycoprotein identification was estimated by periodic acid-Schiff method, and the ribonuclease activity and effect factors were analyzed by agarose gel electrophoresis. Results The electrophoretically pure AmPR-10 was obtained by three-step purification of Q Sepharose Fast Flow, Butyl Sepharose High Performance and SuperdexTM 75 10/300 GL from the crude extraction. The relative molecular weight of AmPR-10 was 16 801. AmPR-10 was highly homologous to PR-10 and has no carbohydrate chains. Incubated at 56 ℃ for 30 min, AmPR-10 exhibited significant ribonuclease activity to total RNA of mammalian cells. The activity was insensitive to NaCl, pH value and mental ions, and weekly inhibited by 0.5 mol/L NaCl, pH 9.0, Mg2+ and Co2+. The activity was the same at EDTA as high as 20 mmol/L. Conclusion The three-step method of exchange chromatography-hydrophobic chromatography-gel filtration chromatography, a stable and rapid separation and purification method of AmPR-10, can be applied for other Chinese herbs. AmPR-10 might play an important role in resistance against virus.