摘要
生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG10 0 0_无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9 2的 15 0 % (W W)PEG10 0 0 17 0 % (W W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W W)PEG10 0 0 17 0 % (W W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分 :卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r min转速的条件下 ,2 0 0min内从鸡蛋清样品中成功分离卵白蛋白 ,其电泳纯度为 10 0 % ,收率为 95 %。
High_speed counte_rcurrent chromatography (HSCCC) is a continuous liquid_liquid partition chromatography without solid matrix, which has the significant features of high resolution and high recovery. The separation of bio_macromolecule in aqueous two_phase systems (ATPs) with HSCCC is still under research, and the establishment of high_speed counter_current aqueous two_phase chromatography (HSCCC_ATP) relies on the improvement of equipment structure and optimization of operation parameters. By using a multi_column high_speed counter_current chromatograph, the separation of protein mixture and the purification of ovalbumin from hen egg white were studied. The effects of pH and PEG concentration on the partition coefficients of proteins were tested in PEG1000_phosphate ATPs, and distinct differences among partition coefficients of proteins were found at pH 9 2 and 15 0% (W/W) PEG concentration in said system. The separation of protein mixture, consisting of cytochrome C, lysozyme and myoglobin was successfully performed in 15 0% (W/W) PEG1000 17 0% (W/W) potassium phosphate ATPs at pH 9 2 with high_speed counter_current chromatograph at rotation speed of 850r/min and flow rate of 0 8mL/min, using upper phase as stationary phase. pH and PEG concentration also had distinct effects on the partition coefficients of the major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme. The optimal pH value and PEG concentration for the purification of ovalbumin by HSCCC_ATP were found to be 9 2 and 16 0% (W/W) respectively. Ovalbumin was successfully purified to homogeneity from the hen egg white sample in 16 0% (W/W) PEG1000 17 0% (W/W) potassium phosphate ATPs at pH 9 2 with high_speed counter_current chromatograph at rotation speed of 850r/min and flow rate of 1 8mL/min, using upper phase as stationary phase. The purification recovery of ovalbumin was around 95%.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第1期129-134,共6页
Chinese Journal of Biotechnology
关键词
高速逆流色谱
双水相
蛋白质
卵白蛋白
分离纯化
high-speed counter-current chromatography, aqueous two-phase system, purification, protein, ovalbumin
