阴阳离子交换色谱串联分离纯化苦瓜籽核糖体失活蛋白

Isolation and Purification of Ribosome-Inactivating Proteins from Bitter Melon Seeds by IonExchange Chromatographic Columns in Series

将阴阳离子交换色谱柱在灌注色谱工作站上串联,使苦瓜籽粗提液中未被阴离子色谱柱吸附的蛋白质直接在阳离子色谱柱上吸附,用盐线性梯度洗脱从阳离子色谱柱上分离出两个具有抗真菌活性的蛋白。两个抗真菌蛋白经鉴定都达到了电泳纯,相对分子质量均为30000左右,N 端氨基酸序列分别为DVSFRLSGADPRSYGMFI与DVNFDLSTATAK,表明所得到的两种蛋白为苦瓜籽中的2个Ⅰ型核糖体失活蛋白α 苦瓜素(α MMC)和β 苦瓜素(β MMC)。抗菌实验表明,α MMC对香蕉枯萎菌和果腐霉菌均具有较强的抑制作用,β MMC对果腐霉菌有较强的抑制作用,而α MMC和β MMC对白绢菌的抑制活性均不明显。采用串联色谱,α MMC和β MMC的质量回收率分别为13 8%和8 0%。

An anion exchange chromatographic column (DEAE-650C) and a cation exchange chromatographic column (CM-650C) were connected in series on a perfusion chromatography workstation. The crude extract of bitter melon seeds flowed through the two columns and the unadsorbed fraction on the DEAE-650C column was then directly readsorbed on the CM-650C column. Two protein components with antifungal activity were eluted from the cation exchange chromatographic column by linear salt gradient. Both of them were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and their relative molecular masses were estimated to be about 30 000. Their N-terminal amino acid sequences are DVSFRLSGADPRSYGMFI and DVNFDLSTATAK. All of the above suggested that they are α-momorcharin (α-MMC) and β-momorcharin (β-MMC), respectively, two typeⅠ ribosome-inactivating proteins (Rips) of bitter melon seeds. α-MMC shows antifungal activity against Fusarium oxysporum and Pythium aphanidermatum, while β-MMC shows antifungal activity against Pythium aphanidermatum. But they are not against Sclerotium rolfsii. In the study, quantitative recoveries of α-MMC and β-MMC were 13.8% and 8.0%, respectively, from decorticated seeds by ion exchange chromatographic columns in series.