不同融合方式表达人全长PDE4B2及其截短突变体的比较

发布时间：2018-05-17 12:52

本文选题：环核苷酸磷酸二酯酶4B2 + 重组表达　；参考：《重庆医科大学》2017年硕士论文

【摘要】：人磷酸二酯酶同工酶4(phosphodiesterase,PDE4)选择性水解cAMP,并分为PDE4A、4B、4C、4D四种亚型。参与炎症反应的单核细胞和中性粒细胞以表达PDE4B2为主。因此,相应的PDE4抑制剂可用于治疗哮喘、慢性阻塞性肺病等炎症相关疾病。磁珠固定化靶蛋白可快速筛选潜在抑制剂混合物,但需要低成本大量表达高活性靶蛋白。前期研究发现,PDE4B2在原核细胞可溶表达丰度低且纯化过程不稳定。有报道显示,现有PDE4B2抑制剂的毒副作用与人全长PDE4B2以咯利普兰(R-rolipram)表征的高亲和力结合构象相关,而治疗作用与以R-rolipram表征的活性位点低亲和力结合构象相关。因此,本论文通过不同融合表达方式对PDE4B2全长及152-528aa截短突变体进行克隆表达纯化及表征,以期获得大量可溶表达的高表观比活且处于以R-rolipram表征的低亲和力结合状态的PDE4B2融合蛋白。将人磷酸二酯酶4B2(hPDE4B2)全长和152-528aa截短突变体融合6His-SUMO在大肠杆菌重组表达,经Ni2+-NTA纯化获得hPDE4B2全长SF-h PDE4B2和截短突变体ST-hPDE4B2重组蛋白。用Western blot检测多肽成分;用偶联碱性磷酸酶的孔雀绿定磷法测定酶活性,表征其km和对模型化合物的抑制常数。结果显示,纯化后的st-hpde4b2纯度较sf-hpde4b2高,st-hpde4b2(0.03u·mg-1)表观比活性接近sf-hpde4b2(1.31u·mg-1)的40倍,活性收率超过100倍;此表达纯化方案总活性收率不高,且westernblot显示均有较多降解片段;测定(r)-rolipram和罂粟碱的抑制常数表明,sf-hpde4b2对(r)-rolipram主要为高亲和力结合构象,而st-hpde4b2主要为低亲和力结合构象。对代表化合物的抑制常数进行数学分析,发现其双对数模型具有单调相关性。因此,与sf-hpde4b2相比,st-hpde4b2用于初步筛选抗炎活性hpde4b2抑制剂可能更有优势。用同源重组的方法将hpde4b2全长和152-528aa截短突变体基因克隆到带有gst标签的载体pgex-6p-1上,表达后经gst亲和层析柱纯化分别获得n-端融合gst的人全长hpde4b2(gf-hpde4b2)和截短突变体(gt-hpde4b2)。然而,纯化后的gf-hpde4b2最大表观比活为0.01u·mg-1,gf-hpde4b2最大表观比活为0.05u·mg-1。gf-hpde4b2和gt-hpde4b2酶活性收率均较低。进一步探索双标签纯化方法,将hpde4b2的n-端和c端与mbp和6-his标签三种形式融合:n-mbp/6his-c、n-6his/mbp-c和n-6his-mbp/6his-c,分别获得mcf-hpde4b2/mct-hpde4b2(带n-mbp/6his-c)、mnf-hpde4b2/mnt-hpde4b2(带n-6his/mbp-c)、mf-hpde4b2/mt-hpde4b2(带n-6his-mbp/6his-c)六种融合蛋白基因质粒,表达后经ni2+-nta和mbp亲和层析纯化获得的全长hpde4b2表观比活性依次是:mcf-hpde4b2(1.11u·mg-1)mf-hpde4b2(0.1U·mg-1)MNF-hPDE4B2(0.01 U·mg-1);截短突变体表观比活性依次是:MCT-hPDE4B2(1.25 U·mg-1)MNF-hPDE4B2(0.6 U·mg-1)MF-hPDE4B2(0.3 U·mg-1);全长hPDE4B2和截短突变体重组蛋白的活性收率也显著高于前两种方案。鉴于测定PDE4活性筛选抑制剂对靶蛋白活性的要求,以MCF-h PDE4B2和MCT-hPDE4B2为靶蛋白在成本上显得更有优势。综上所述,本研究成功获得hPDE4B2全长和截短突变体与三种不同标签的融合表达重组蛋白:融合6His-SUMO标签的hPDE4B2截短突变体的活性收率效果较hPDE4B2全长好;融合GST标签活性收率效果均不理想;N端融合MBP标签C端融合6his标签的hPDE4B2全长和截短突变体融合表达及纯化方案的活性收率最好。因此,推荐高通量初筛潜在抗炎活性的PDE4抑制剂用MCT-hPDE4B2,确认潜在抗炎活性的PDE4抑制剂用MCF-hPDE4B2。
[Abstract]:The human phosphodiesterase isoenzyme 4 (phosphodiesterase, PDE4) selectively hydrolyzed cAMP and divided into four subtypes of PDE4A, 4B, 4C, 4D. The monocytes and neutrophils involved in the inflammatory response expressed PDE4B2. Therefore, the corresponding PDE4 inhibitors can be used in the treatment of asthma, chronic obstructive pulmonary disease and other inflammatory related diseases. Magnetic beads immobilized target protein The potential inhibitor mixture can be quickly screened, but high activity target proteins are needed at low cost. Previous studies have found that PDE4B2 has low soluble expression in prokaryotic cells and is unstable in purification process. It is reported that the toxic side effects of existing PDE4B2 inhibitors are associated with the high affinity of human full-length PDE4B2 with the high affinity of R-rolipram. Conformation is related, and the therapeutic effect is related to the conformation of low affinity binding to the active site characterized by R-rolipram. Therefore, the whole length of PDE4B2 and the truncated 152-528aa mutants were cloned and characterized by different fusion expressions, in order to obtain a large amount of soluble expressed high apparent specific activity and be characterized by R-rolipram. PDE4B2 fusion protein of low affinity binding state. Recombinant human phosphodiesterase 4B2 (hPDE4B2) full length and 152-528aa truncated mutants were fused 6His-SUMO in Escherichia coli, hPDE4B2 full SF-h PDE4B2 and truncated mutant ST-hPDE4B2 recombinant protein was purified by Ni2+-NTA. The polypeptide components were detected by Western blot. The enzyme activity of malachite green and phosphorus was determined to characterize its km and the inhibition constant to the model compound. The results showed that the purity of purified st-hpde4b2 was higher than that of sf-hpde4b2, and the apparent specific activity of st-hpde4b2 (0.03u. Mg-1) was 40 times that of sf-hpde4b2 (1.31u. Mg-1), and the yield was over 100 times, and the total yield of the expression and purification scheme was not high, and Wester was not high, and Wester was not high. Nblot showed that there were more degradation fragments, and the inhibition constants of (R) -rolipram and papaverine showed that sf-hpde4b2 to (R) -rolipram was mainly a high affinity binding conformation, while st-hpde4b2 was mainly a low affinity binding conformation. The mathematical analysis of the inhibition constants for the representative compounds showed that the double logarithmic model had a monotone correlation. Therefore, Compared with sf-hpde4b2, st-hpde4b2 may be more advantageous for the preliminary screening of anti inflammatory hpde4b2 inhibitors. The whole length of hpde4b2 and the 152-528aa truncated mutant gene were cloned to the carrier pgex-6p-1 with the GST label using the homologous recombination method, and the whole length hpde4b2 (gf-) of the n- terminal fusion GST after the expression of the GST affinity chromatography column was expressed after the expression of the GST affinity chromatography column. Hpde4b2) and truncated mutants (gt-hpde4b2). However, the maximum apparent ratio of the purified gf-hpde4b2 is 0.01u. Mg-1, and the maximum apparent ratio of gf-hpde4b2 to the activity of 0.05u. Mg-1.gf-hpde4b2 and gt-hpde4b2 enzyme is lower. Further explore the double label purification method, which combines the n- and C ends of hpde4b2 with the three forms: 6his-c, n-6his/mbp-c and n-6his-mbp/6his-c, respectively obtained mcf-hpde4b2/mct-hpde4b2 (n-mbp/6his-c), mnf-hpde4b2/mnt-hpde4b2 (n-6his/mbp-c), mf-hpde4b2/mt-hpde4b2 (n-6his-mbp/6his-c) six fusion protein gene plasmids, after expression by ni2+-nta and MBP affinity chromatography, the full length hpde4b2 apparent specific activity is in turn: Mcf-hpde4b2 (1.11u. Mg-1) mf-hpde4b2 (0.1U. Mg-1) MNF-hPDE4B2 (0.01 U. Mg-1); the apparent specific activity of the truncated body is as follows: MCT-hPDE4B2 (1.25 U mg-1). The yield of egg white in the whole length and the truncated body weight group is significantly higher than the first two schemes. The requirements for the activity of target proteins by selective inhibitors, MCF-h PDE4B2 and MCT-hPDE4B2 as target proteins are more advantageous in cost. To sum up, this study successfully obtained the fusion expression of hPDE4B2 full length and truncated mutants and three different labels: the activity yield of hPDE4B2 truncated mutant with 6His-SUMO tag is more effective than hPD The full length of E4B2 is good; the effect of the fusion GST tag active yield is not ideal; the activity yield of the hPDE4B2 full length and the truncated mutant of the N terminal fusion MBP tag C terminal fusion 6His tag is the best. Therefore, the PDE4 inhibitor which recommends the potential anti-inflammatory activity of the high throughput screening is used to confirm the PDE4 inhibitor for the potential anti-inflammatory activity. Using MCF-hPDE4B2.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R3411

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