用液相等电聚焦电泳纯化藻蓝蛋白亚基

以纯藻蓝蛋白(C-phycocyanin, C-PC)为材料, 采用Rotofor系统进行液相等电聚焦(Liquid-phase isoeletric focusing, LP-IEF)电泳纯化C-PC的α, β亚基, 探讨蛋白质亚基纯化的制备电泳(Preparative eletrophoresis)技术. 结果显示, 样品经2次等电聚焦电泳后, C-PC 的α, β亚基分别浓集在pH=4.9和pH=4.1附近, 平板超薄等电聚焦(Slab ultra thin IEF)和SDS-PAGE电泳鉴定表明分别为高纯度的C-PC α, β亚基. 提示LP-IEF是分离纯化等电点差异蛋白质活性亚基的简便有效的方法.

Purification and Preparation of Active Subunits of C-phycocyanin by a Protocol of Liquid-phase Isoeletric Focusing

A process of liquid phase isoeletric focusing(LP-IEF) was developed and conducted in a 8.0 mol/L urea medium in Rotofor system for isolation, purification and preparation the α and β subunits of C-PC(αβ)3 purified from Spirulina platensis. After two steps of pre- and re- fractionating, collected fractions with α or β subunit were identified and characterized by SDS PAGE and slab ultra thin IEF. Its absorption and fluorescence spectra were determined. The results show that the α and β subunits were concentrated at about pH=4.9 and pH=4.1 respectively and the total recovery of α and β subunits was 392%. The α and β subunits of C-PC with a high purity as well as activity maintaining were obtained easily by this LP-IEF protocol, suggesting that it should be an ordinary but effective method for purification and preparation of protein subunits with different pI values.