3',5'-二磷酸核苷酸酶在亲和双水相胶束系统中分配行为研究  

Studies on partitioning behavior of 3',5'-bisphosphate nucleotidase in affinity-based aqueous two-phase micellar system

作　　者：刘耀[1] 周鑫鑫[1] 邓刚[1] 

机构地区：[1]浙江师范大学化学与生命科学学院,浙江金华321004

出　　处：《工业微生物》2017年第4期43-48,共6页Industrial Microbiology

基　　金：国家自然科学基金(No.21006098)

摘　　要：以高分子表面活性剂HM-EO为主成相剂,金属螯合表面活性剂Triton X-114-IDA-Cu(Ⅱ)(TX-Cu(Ⅱ))为辅成相剂,构建新型亲和双水相胶束系统(ATPMS)以提高目标产物的萃取选择性,并考察重组蛋白3',5'-二磷酸核苷酸酶(YND)在系统中分配行为。结果表明,系统中不含亲和配基时YND主要分配于胶束缺失相;随着亲和配基含量的增加,YND与TX-Cu(Ⅱ)亲和结合而逐渐分配到胶束富集相并且在系统中显示出优异的稳定性;调节溶液p H能够影响YND亲和分配,最适萃取条件为pH 9.0;增大无机盐浓度,导致更多杂蛋白分配到胶束缺失相,然而对YND分配影响较小。在2.5%HM-EO、0.125%TX-Cu(Ⅱ)、p H 9.0、50 mmol/L Na Cl条件下,实验获得65.8%的酶活回收率。因此亲和ATPMS可以有效用于对富组氨酸蛋白YND的分离纯化,为该体系在重组蛋白的分离纯化试验提供相应的基础依据。In order to improve the specificity of ATPMS,a novel affinity-based aqueous two-phase micellar system（ ATPMS） was constructed by introducing a metal-chelated surfactant Triton X-114-IDA-Cu（ Ⅱ）（ TX-Cu（ Ⅱ）） into an aqueous solution of hydrophobically modified ethylene oxide polymer（ HM-EO）,and the possibility of this HM-EO/TXCu（ Ⅱ） system for separating histidine-tagged proteins was investigated by the affinity partitioning of recombinant protein3＇,5＇-bisphosphate nucleotidase（ YND）. It was found that in the absence of TX-Cu（ Ⅱ）,the YND tended to partition into the micelle-poor phase,where it experienced fewer excluded-volume effects. However,with the addition of TX-Cu（ Ⅱ）,the YND was partitioned preferentially to the micelle-rich phase due to affinity binding and was stable in the system. The p H of the solution could affect the partitioning behavior of YND,and the optimum condition for the extraction of YND was pH 9. 0. With increasing Na Cl concentration,more protein impurities were removed to the micelle-poor phase,but the partition coefficient of YND fluctuated slightly. Finally,the YND was attained in the micelle-rich phase with a recovery yield of 65. 8% under the conditions of HM-EO 2. 5%,TX-Cu（ Ⅱ） 0. 125%,p H 9. 0 and 50 mmol/L Na Cl. The results indicated that the new affinity-based HM-EO/TX-Cu（ Ⅱ） system had high partitioning performance which was promising for effectively separation of the histidine-tagged proteins.

关 键 词：双水相胶束系统 金属螯合表面活性剂 亲和分配 3' 5'-二磷酸核苷酸酶 

分 类 号：Q55[生物学—生物化学]