The Purification, Physico-chemical Character And Bioactivity Of 66kDa Activator Protein From Alternaria Tenuissima

In this research, a 66kDa activator protein was extracted and purified from the Alternaria tenuissima. The silver staining showed a single band, which verified it is purity. This protein biological activity, physical character, glycoprotein identification and MS analysis were executed to make theoretical foundation for further research on the function mechanism of this protein.1 The extract and purification of 66kDa activator protein from Alternaria tenuissima The suitable buffer for extraction and conservation of this protein was screened from Good buffer system. The most nucleic acid in the extract was removed by protamine. Pigment was effectually absorbed by active carbon. The primary fractionation extract was done by ethanol precipitation, and the following purification was executed by DEAE anion exchange chromatography. After desalting by HiprepTM Desalting column and concentrated by ultrafiltration, a single band was detected by SDS-PAGE and silver staining.2 The 66kDa activator protein physical and chemical charactersThe apparent molecular weight of this protein was 66kDa on SDS-PAGE. The PI of this protein was about 4.27 on 2D-electrophoresis. The protein was treated by 100℃boiling bathe and then cooled down to room temperature. The SDS-PAGE detection of the treated protein verified this protein was a thermostable protein.3 The 66kDa activator protein glycoprotein identificationThe glycoprotein specific staining was done with horseradish peroxidase as positive control and soybean trypsin as negative control, and the result showed 66kDa protein was a glycoprotein.4 The 66kDa activator protein biological activity determinationThe TMV resistance assay was respectively done by treating tobacum Samsun with 4μg/mL,6μg/mL,8μg/mL protein solution and mechanical inoculation after 72 hour. The result was that the 8μg/mL protein solution had a necrosis inhibit rate at 49%. Wheat seeds were also treated by 8μg/mL and then cultured respectively at room temperature and low temperature of 15℃. The average plant height detection was done after 5 days and the treatment and control at room temperature were 9.87cm and 9.82cm, which had no obvious difference. But the treatment and control at low temperature of 15℃were 6.89cm and 4.7cm, which demonstrated the protein have a notable effect on wheat cold resistance.5 The 66kDa activator protein MS analysisBy MS analysis of purified protein after enzyme digestion, more than 10 relative peptide information was gained. After comparison with database by MASCOT, it was found that this protein has partly homology with Protein disulfide isomerase(PDI) from Alternaria alternata.