节点文献
N-乙酰-D-神经氨酸的酶法合成和正选择表达载体的构建
【作者】 朱宇鹏；
【导师】 尚广东；
【作者基本信息】 南京师范大学 ， 生物技术， 2011， 硕士
【摘要】 N-乙酰-D-神经氨酸是化学合成扎那米韦的关键中间体,也是合成其他抗流感药物的主要原料。目前,N-乙酰-D-神经氨酸主要通过酶催化法制备。首先由N-酰基-D-葡萄糖胺2-异构酶将N-乙酰-D-葡萄糖胺异构化为N-乙酰-D-甘露糖胺；从后者到N-乙酰-D-神经氨酸有两个途径：一种是神经氨酸醛缩酶催化N-乙酰-D-甘露糖胺与丙酮酸之间的缩合而得到N-乙酰-D-神经氨酸；另一种是神经氨酸合成酶催化N-乙酰-D-甘露糖胺与磷酸烯醇式丙酮酸缩合为N-乙酰-D-神经氨酸。本研究通过同源搜索找到与已知神经氨酸合成酶具有较高同源性的蛋白,进而在大肠杆菌中进行克隆、表达和纯化。同时测定了不同来源的神经氨酸醛缩酶、神经氨酸合成酶以及N-酰基-D-葡萄糖胺2-异构酶的活性。结果表明来源于大肠杆菌的醛缩酶活性最高,以全细胞进行催化,37℃下反应1h后,神经氨酸的摩尔收率最高可以达到23.31%；来源于脑膜炎双球菌的神经氨酸合成酶在全细胞水平具有很高的合成酶活性,37℃下全细胞催化反应30分钟后Neu5Ac的摩尔收率最高可以达到50.96%；来源于多拟形杆菌的BT0453蛋白的异构酶活性较好,与大肠杆菌醛缩酶偶联反应,反应时间为4h后,神经氨酸的摩尔收率最高达到2.77%。目前将来源自大肠杆菌的三种伴侣蛋白作为融合标签构建了伴侣蛋白融合表达载体。选取A113695和RB3348与伴侣蛋白基因融合表达。结果表明,目的基因与三种伴侣蛋白融合表达的重组蛋白可溶性明显增加,启动因子作为融合标签的融合蛋白表达量最高。BandScan软件分析表明,a113695基因与启动因子基因融合表达时,表达出95.5KD融合蛋白,所表达的融合蛋白几乎均为可溶性蛋白,其可溶性蛋白的表达量占总表达蛋白的比例为37.2%,高于a113695单独表达时的7.1%的比例；RB3348基因与启动因子基因融合表达时,可溶性蛋白的表达量占总表达蛋白的比例为53.0%,远高于单独表达时4.4%的比例。正选择克隆载体是一类通过抗生素或化合物对重组克隆进行直接筛选的载体,最大优点是其可保证很高的克隆效率,有时可达100%。有报道显示β-内酰胺酶基因(bla)在缺失C端密码子(290位色氨酸密码子)时,将不能很好的表达氨苄青霉素抗性功能；而定向插入外源基因后将重建bla的C端密码子,进而使β-内酰胺酶重获氨苄青霉素抗性功能。本研究将色氨酸缺失的β-内酰胺酶基因克隆至带有TEV蛋白酶酶切位点的表达载体上,构建了正选择表达载体pPAE以及pPAE-MBP。选取大肠杆菌醛缩酶nanA和人源肾素结合蛋白hRnBP做克隆范例。实验结果证明pPAE和pPAE-MBP的克隆效率均达100%,同时所克隆的基因成功在大肠杆菌中表达。
【Abstract】 N-Acetyl-D-neuraminic acid (Neu5Ac) is the key intermediate to chemical synthesis of Zanamivir, one of the two medicines used in the treatment of influenza. N-acetyl-D-neuraminic acid is currently made through enzymatic synthesis; the procedure consists of two steps:first, N-acetyl-D-glucosamine 2-epimerase (AGE) catalyzes the transformation of N-acetyl-D-glucosamine (GlcNAc) into N-acetyl-D-mannosamine (ManNAc), after which two pathways exist. One is the condensation of ManNAc and pyruvate into Neu5Ac through aldolase, and another is the condensation of ManNAc and phosphoenolpyruvate through synthetase.Through homology search, we found several proteins that have identity with N-acetyl-D-neuraminic acid synthetase (neuB) from E. coli K1. These proteins were cloned, expressed and purified. The activities of aldolase, NeuB and AGE from different sources were determined. The results show that E. coli aldolase has the best activity:after 1 hour incubation at 37℃, the yield of Neu5Ac from ManNAc was 23.31% in whole-cell catalysis. NeuB from Neisseria meningitidis showed the best synthetase activity, with a 50.96% molar conversion after 30 minutes incubation at 37℃in whole-cell catalysis. BT0453 from Bacteroides thetaiotaomicron VPI-5482 showed the best AGE activity in that when coupled with E. coli aldolase in whole-cell catalysis,2.77% molar conversion from GlcNAc was observed after 4 hours incubation at 37℃in whole-cell catalysis.Three E. coli chaperones were used as fusion tag to contruct chaperone-fusion protein expression vectors. Two proteins, A113695 and RB3348, were expressed in these vectors. Greatly improved solubility was observed for the expression of fusion proteins compared with the untagged protein expression. The trigger factor fused proteins showed the largest expression level among the chaperones. Bandscan analysis indicated that A113695 fused with trigger factor was a 95.5KD fusion protein, which was almost completely soluble, and the percentage of soluble fusion-protein in the total protein is 37.2%, higher than the percentage of untagged protein (7.1%). RB3348 fused with trigger factor was almost completely soluble too, and the percentage of soluble fusion-protein in the total protein is 53.0%, much higher than the percentage of untagged protein (4.4%).The positive-selection vector is one kind of vectors that directly selecte recombinant clones through antibiotics or chemical compounds. The biggest advantage is that the cloning efficiency that can be up to 100%. It was reported that bla missed the C-terminal W290 codon lost its ampicillin-resistance activity, while cloning a target gene carrying the respective sequence to complement this amino acid residue at the C-terminus of Bla into the vector would result in restoring the protein’s function. In this report, we construced improved positive-selection protein expression vectors pPAE and pPAE-MBP by cloning Bla that lacks of several C-terminus amino acids into an expression vector that harboring the restriction site of TEV protease. Cloning experiments with E. coli aldolase gene (nanA) and human renin binding protein (hPnBP) showed that both pPAE and pPAE-MBP have 100% cloning efficiency, and the genes successfully expressed in E. coli.