亲和层析法纯化磷酸激酶的研究ⅠP349353.pdfVIP

亲和层析法纯化磷酸激酶的研究(I) 应国清王玉姣 (浙江工业大学药学院，浙江杭州310014) 摘要：为确定一条采用亲和层析技术分离纯化酵母中磷酸激酶的较佳工艺路线， 采用分步盐析初步纯化己糖激酶(HK)，考察硫酸铵饱和度对黼析效果的影响； 以ATP为配基，SESA活化纸纤维索制备亲和层析剂，确定最佳偶联条件和最佳 静态吸附pH值后，将粗酶液直接上亲和层析柱，看分离纯化的效果；在载体和 配基之间接上1,6．己二胺为间隔臂，比较接臂前后对吸附选择性的影响。粗酶液 经分步盐析后纯化倍数达3．5倍；粗酶液直接上亲和层析柱，一步纯化后纯化倍 数达3．7倍：并确定了接间隔臂可以提高载体对HK的选择性。实验证明：以ATP 为配基的亲和层析法纯化酵母细胞中的磷酸激酶是可行的。 关键词：亲和层析，磷酸激酶，纯化 PurificationofKinase byAmnity Chromatography YingGuo-Qing，WangYu-jiao ofPharmaceutical Zhou Science，Zhe (College JiangUniversityofTechnology,Hang 310014，China) Tosearchasuitable routeof ofkinasein Abstract process purification yeastby preparatorilypurifiedbysalt affinitychromatography,hexokinase(HK)was the conditionand confirmingoptimizedcoupling absorbingpH，the preciptation．After crude was affinity mediumcombinedwithATP enzymepurifiedby chromatographic SESAassurfactant．Thearrfl was andwith space 1,6-Hexanediaminecoupled to absorbtioneffectwithoutthe betweenthematrixandthe the ligandcompare space arm．Thecrude was 3．5fold fractionalsalt enzymepurified by preciptation．3．7fold betterwith of the was byone—stepaffinitychramatography,andselectivity coupling arln．It to thekinasein the isfeasible affinity