Purification of alpha-sarcin and an antifungal protein from mold (Aspergillus giganteus) by chitin affinity chromatography.
A simple method for preparation of alpha-sarcin and an antifungal protein (AFP) from mold (Aspergillus giganteus MDH 18894) has been developed. alpha-Sarcin and AFP were purified simultaneously by chitin affinity column chromatography and gel filtration. By this method, 4.5 mg of pure alpha-sarcin and 6.9 mg of pure AFP were obtained from 2 liters of culture medium. Compared with other purification methods such as ion-exchange column chromatography, this procedure was very simple and specific. The purified alpha-sarcin and AFP were homogeneous as characterized by SDS-polyacrylamide gel electrophoresis. Both alpha-sarcin and AFP exhibited the binding activity to generated chitin. Soluble glycochitin decreased the intensity of fluorescence of alpha-sarcin and made the lambda(em)m shift from 340 to 347 nm. Titration of alpha-sarcin with N-bromosuccinimide under native conditions revealed that two tryptophans (Trps) were all located in the core part of alpha-sarcin molecule. This indicated that Trps were not involved in the binding of alpha-sarcin to chitin. Glycochitin in the culture medium increased the expression of alpha-sarcin, while it had no effect on the expression of AFP. Unlike other ligands such as Cibacron blue for the affinity purification of alpha-sarcin and AFP, glycochitin increased the nuclease activity of alpha-sarcin.
Liu
Ren-shui
RS
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
Huang
Hu
H
Yang
Qiang
Q
Liu
Wang-Yi
WY
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001230:Aspergillus / Q000378:metabolism*; D001974:Bromosuccinimide / Q000737:chemistry; D002474:Cell-Free System; D002686:Chitin / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification*; D005656:Fungal Proteins; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000935:Antifungal Agents; D005656:Fungal Proteins; D002686:Chitin; C014801:alpha-sarcin; D004722:Endoribonucleases; D012260:Ribonucleases; D001974:Bromosuccinimide