The Purification And ESI-MS Analysis Of A Glycosylated Activator Protein From Alternaria Tenuissima

The Activator Proteins extracted from Alternaria tenuissima can enhance plant growth and induce disease resistance and show three bands on SDS-PAGE gel, 33kD, 42kD and 66kD, of which 66kD has been proved to be a glycosylated protein. To further study its characteristics and action mechasim, this research separates and purifies this glycosylated protein by chromatography and other techniques and obtains a pure protein showing a single band on the silver-stained SDS-PAGE gel. The O-linked glycans are removed by TFMS chemical method and the glycosylation sites are analysed. Based on the standard protein HPLC-ESI-MS analytical technique, the glycosylated activator protein HPLC-ESI-MS analytical technique is built, and the accurate molecular weight of the glycosylated protein is measured. The MS data search in the protein database shows that this glycosylated protein may be a new protein.The concentration methods of ammonium sulphate fractionation and cold alcohol precipitation, are used to concentrate the proteins and remove the polysaccharide, which influences the protein purification. The buffer and elution conditions are optimized and the glycosylated activator protein is then purified by anion exchange chromatography and size exclusion chromatography. The result shows that the MES buffer at the pH6.0, which contained 0.2M NaCl, could purify the protein. The elution of the second peak after size exclusion chromatography shows a single band on the silver-stained SDS-PAGE gel. The pI=4.27 of the purified glycosylated activator protein is acquired by liquid isoelectric focusing electrophoresis.The O-linked glycans of the glycosylated activator protein are removed by TFMS chemical method and the protein shows two bands on the SDS-PAGE gel, which means this glycosylated activator protein may have at least two glycosylation sites.The standard protein lysozyme and BSA are used to build a HPLC-ESI-MS protein analytical technique. The accurate molecular weight of the lysozyme is measured at 14310.7 dalton. The trypsin digest peptides of BSA are analysed by RP-HPLC-ESI-MS and obtains an accurate identification by MASCOT search.Based on the standard protein HPLC-ESI-MS analytical technique, the glycosylated protein HPLC-ESI-MS analytical technique is built, and the accurate molecular weight of the glycosylated protein is measured at 50270 dalton. The trypsin digest peptides of the glycosylated activator protein are analyzed by RP-HPLC-ESI-MS and no high score matches are found in MASCOT search, which means this glycosylated protein may be a new protein and needs further study to be proved.