用液相等电聚焦电泳纯化藻蓝蛋白亚基

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

以纯藻蓝蛋白(C-phycocyan in,C-PC)为材料,采用Rotofor系统进行液相等电聚焦(L iqu id-phase iso-eletric 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 eletro-phoresis)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 u ltra th in IEF)和SDS-PAGE电泳鉴定表明分别为高纯度的C-PCα,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.

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 platertsis. After two steps of preand 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 a and fl subunits was 39.2%. The a and fl subunits of C-PC with a high purity as well as activity maintaining were obtained easily by this LP-IEF protocol, sugges- ting that it should be an ordinary but effective method for purification and preparation of protein subunits with different pI values.