Isolation, Purification And Structural Analysis Of Plant Activator Protein

With Alternaria spp., plant activator protein was distilled, separated and purified, and then the properties so identified. For the study of the structures and functions of glycoprotein. This experiment provided a theoretical evidence for the activator protein and its function mechanism. Results as follows.1. Through the concussive culture of mycelium at 28℃of 72 hours, the yield of 66KD protein reached the highest final level. When distilled by 70%, 80% ethanol, the band of 66KD was strongest, which indicated 66KD protein was an inorganic protein. When deposited by 20%～30% ammonia sulfate, deposition of total proteins reached the highest final level, about 43.23% of total proteins. Result of two-dimensional electrophoresis indicated the pI of activators were focused on acidic regions.2. By molecular weight distribution ion exchange chromatograph(HitrapTM DEAE) , two proteins which had bioactivity were gained in separating peak under conditions of pH 3.5, pH4.0 acetate acidic buffer, with the molecular weight of 66KD and 45KD. Gradient separating had a higher extent of purification with the disadvantage of longer time. 66KD protein was elementally purified with HitrapTM 26/10 desalting column to remove salt, small molecule impurities and degradabilities. Then Hitrap Superdex 75/300 was used for the further purification .3. Detection of glycoprotein. The purity and molecular weight were detected by 12%Native-PAGE and 12%SDS-PAGE. 66KD and 45KD proteins after purification were pure by silver staining. Special staining indicated 66KD protein was a glycoprotein and its characteristic absorption peak was 257nm. The ratio of sugar and protein was 1.75. It was O-glycosidic linkage differentiated by PNGase F enzyme andβ-elimination reaction. The glycoprotein was sensitive to pepsin not to pancreation. It contained 53KD and 10KD proteins after being cut with pepsin.4. Detection of glycoprotein odd-shapes. It was detected by 12%SDS-PAGE, recycling and isoelectric focusing electrophoresis. Result indicated it had clearly three types of odd-shapes when being shakingly cultured after 48 hours, and pIs were 3.5, 3.7 and 4.3, respectively. The result was identical to that in HitrapTM DEAE.5. The analysis and searches of peptide mass of fingerprint in Mascot indicated there were no matching proteins, a new glycoprotein was possible.6. Structural analysis of glycoprotein. After dissolving in three solutions as CH3OH:H2O=1:1(V/V),CH3OH:H2O=1:20(V/V) and H2O, and then being scanned in circular dichroism spectra with the wavelength from 190nm to 240nm, the results included helix, beta, turn and random. After perturbation with CH3OH, content of helix was kept 15.8%, while beta content increased from 46.1% to 48.6%. Content of turn decreased from 13.5% to 6.2% while random content increased when organic phase transformed to watery phase.7. Search and detection of protein crystals and its growing condition. 98 solutions of Crystal Screen TM HR2-110 and Crystal Screen TM HR2-112 were used to find out a suitable condition for crystal growing. As a result, glycoprotein crystals could get under 14th buffer in Crystal Screen TM HR2-110 at 16℃. But we did not receive perfect data because of limited XRD spots.8. Determination of bioactivity of glycoprotein. With the seeds soaked in glycoprotein solution for 6 hours, the growth status of seed germination after 2 days and the height of wheat after 5 days was determined. Results were as follows: The grow status of seed germination in treated terms were 93.62%, 97.87% and 93.61%, respectively, higher than 87.15% of CK. The height of wheat increased after being treated with different concentrations of glycoprotein. The correlation coefficients indicated the differences were all significant.