Cbl-b蛋白的重组表达、纯化和活性检测方法的建立

Cbl-b是一种重要的泛素连接酶E3酶,负责与多种底物蛋白的特异识别,并因此介导特定蛋白的降解。研究表明,Cbl-b缺失可激活自然杀伤细胞,从而阻止肿瘤的扩散转移,因此Cbl-b被认为是一种肿瘤免疫治疗的新靶点。但目前针对Cbl-b蛋白的生化性质以及体外活力测定方法研究较少。本研究构建了Cbl-b不同结构域的重组表达载体。通过对其表达和纯化条件进行优化,确定了Cbl-b的底物结合功能域的最优表达条件,即使用感受态BL21(DE3),在IPTG浓度为0.5 mmol/L、温度25℃下诱导10 h。利用亲和柱和分子筛层析联用纯化方法,提纯得到大小为65 kD的GST-Cbl-b(39~368)重组蛋白,其纯度可达95%。进一步,通过药物设计方法,设计并合成带有荧光素标记的Cbl-b底物Cblin-FAM,并利用配体结合实验验证了Cbl-b(39~368)结合底物的活力。本研究得到了具有活力的Cbl-b底物结合功能域并建立了其体外表达纯化方法,为后续发展基于Cbl-b纯酶的体外泛素化检测方法,以及发展新型、特异靶向Cbl-bE3酶的调控物提供了物质基础。

Recombinant Expression and Purification of Cbl-b Protein and Establishment of Activity Detection Method

Cbl-b is an important ubiquitin ligase E3,which is in charge of the recognition of various types of substrates and thus specifically mediates their degradations.Studies have shown that the knockout of Cbl-b gene has been proved to activate the natural killer cells,and subsequently prevent the spread of tumor metastasis.Thus,Cbl-b is throught to be a new target for tumor immunotherapy.However,there were few studies on the biochemical characters and assays for Cbl-b were less explored,since there are few in vitro biochemical studies foractivity of Cbl-b protein.In this study,we constructed the recombinant expression vectors containing theof different domains of Cbl-b were constructed.protein using prokaryotic expression system.After the optimization of the expression and purification conditions for Cbl-b,the optimal expression conditions of substrate binding domain of Cbl-b were determined,we found that Cbl-b(39~368),a substrate binding domain of Cbl-b,could be using receptive BL21(DE3),induced for 10 h at IPTG concentration of 0.5 mmol/L,at 25 substantially expressed in BL21(DE3)competent cells under the condition of 0.5 mmol/L IPTG,25℃.and an induction time of 10 h.Using the combination method of affinity chromatography and molecular sievessize exclusion chromatography,the recombinant GST-Cbl-b(39~368),was purified with a molecular weight of 65 kD and purity of 95%.Furthermore,we designed and synthesized a fluorescein-labeled substrate of Cbl-b with the drug design approach,i.e.Cblin-FAM through the drug design method,and the activity ofused it to set up a ligand-binding assay for Cbl-b(39~368)in combination with the substrate was verified by the ligand binding assay.The assay was used to test the substrate binding activity of Cbl-b(39~368).Overall,weThis study found the optimal expression conditions foractive Cbl-b substrate binding functional domainprotein,and constructed a biochemical assay for detection the in vitro activity of Cbl-b.These findings,wihch could serve asprovied a material basis for the subsequent development ofnot only for reconstituting the in vitro ubiquitination mediated bybased on Cbl-b pure enzyme and the development of a new type of regulator specifically targetingbut also for identification of specific inhibitors for Cbl-b E3 ligase.