嘌呤核苷磷酸化酶基因的克隆及原核表达载体的构建

通过PCR方法从产气肠杆菌、胡萝卜软腐欧文氏菌、大肠杆菌扩增嘌呤核苷磷酸化酶(PNPase)基因,然后将扩增的约720bp的基因片段克隆到pET-28b表达载体上,构建重组PNPase的表达载体。核苷酸及推导的氨基酸序列分析表明,该基因在三个菌株之间有很高的同源性。SDS-PAGE电泳结果显示出明显的特异性蛋白质条带,其分子量约为29.8kDa.该载体的构建为进一步研究核苷及其类似物的生物合成奠定基础。     

relA基因在重组大肠杆菌Bk21合成嘌呤核苷磷酸化酶作用研究

利用Red同源重组技术敲除大肠杆菌BL21中relA基因,获得ArelA突变株。进一步研究表明在LB复合培养中reIA基因对大肠杆菌的生长几乎没有明显影响,但是对其重组蛋白的合成有着较大的影响：降低了25％。结果表明,relA基因在大肠杆菌表达重组蛋白方面有着较重要的作用。     

relA基因在重组大肠杆菌BL21合成嘌呤核苷磷酸化酶作用研究

利用Red同源重组技术敲除大肠杆菌BL21中relA基因,获得△relA突变株。进一步研究表明在LB复合培养中relA基因对大肠杆菌的生长几乎没有明显影响,但是对其重组蛋白的合成有着较大的影响:降低了25%。结果表明,relA基因在大肠杆菌表达重组蛋白方面有着较重要的作用。     

嘌呤核苷磷酸化酶与嘧啶核苷磷酸化酶的融合表达及酶法合成嘌呤核苷类产物

将嘌呤核苷磷酸化酶与嘧啶核苷磷酸化酶进行融合,提高生物酶法催化合成克拉屈滨等嘌呤核苷类产物的产率.设计不同的刚性、柔性连接短肽(Linker),将嘧啶核苷磷酸化酶EcUP与嘌呤核苷磷酸化酶AmPNP连接融合,考察酶融合蛋白的可溶性表达与活性情况.使用EEEEEEKKK短肽连接的融合蛋白EcUP-L4-AmPNP可溶性表...     

大肠埃希菌嘌呤核苷磷酸化酶基因载体的构建及对胰腺癌细胞的作用

目的构建大肠埃希菌（Escherichia coli）嘌呤核苷磷酸化酶（purine nucleoside phosphorylase,PNP）基因表达载体,研究其生物活性,为肿瘤的基因治疗奠定基础。方法PCR扩增大肠埃希菌K12的PNP基因,T4连接酶将PNP连接人pMSCV逆转录病毒载体,构建重组逆转录病毒载体pMSCV／PNP。pM—SCV／PNP转化感受态大肠埃希菌XLI-Blue,提取pMSCV／PNP,酶切、PCR和测序鉴定。病毒包装细胞293产生重组逆转录病毒pMSCV／PNP,流式细胞仪测病毒滴度。pMSCV／PNP转染胰腺癌细胞BXPC-3,倒置荧光显微镜观察,FACS分离转染阳性细胞（GFP阳性）。RT—PCR检测PNPmRNA在胰腺癌细胞BXPC-3细胞中的表达,MTT法检测PNP基因的生物活性。结果PCR扩增出大肠埃希菌PNP基因（738bp）,酶切和PCR的电泳条带显示pMSCV／PNP,测序结果正常。293包装细胞产生高滴度（3．6×10^7U／m1）重组逆转录病毒pMSCV／PNP。RT—PCR实验结果表明,pMSCV／PNP转染的胰腺癌细胞BXPC-3表达PNPmRNA。前药6-甲基嘌呤-2’-脱氧核苷（MePdR）作用72h浓度达1．00mg／L,BXPC-3／PNP细胞存活率为10．09％,随着MePdR浓度加大,BXPC-3／PNP细胞存活率继续下降直至为0。结论构建了pMSCV／PNP载体,获得了表达大肠埃希菌PNP基因的BXPC-3细胞克隆,PNP／MePdR自杀基因系统对胰腺癌细胞BXPC-3有较强的抑杀作用。     

假交替单胞菌XM2107嘌呤核苷磷酸化酶基因克隆表达、重组蛋白纯化及酶学性质

【目的】嘌呤核苷磷酸化酶(PNP,EC.2.4.2.1)在酶法合成核苷类药物及中间体中具有广泛应用。本文研究的目标是,获得极地嗜冷菌假交替单胞菌Pseudoa lteromonas sp.XM2107嘌呤核苷磷酸化酶编码基因,并对该酶酶学性质进行研究,以考察该酶在核苷类中间体及药物合成中的潜在应用价值。【方法】利用同源序列PCR技术从Pseudoa lteromonas sp.XM2107基因组DNA中扩增出其编码嘌呤核苷磷酸化酶基因,测序获得编码序列。将该基因在大肠杆菌BL21(DE3)中进行重组表达以及金属螯合层析纯化,对其酶学性质进行初步研究。【结果】经过测序获得了该酶编码基因序列,全长702 bp,共编码233个氨基酸,大小为25 kDa,Genbank登录号为GQ475485。酶学性质研究发现,该重组酶最适反应温度为50℃,最适酶促反应pH为7.6(25 mmol/L磷酸盐缓冲液),最适酶促反应底物为肌苷(Km值0.389 mmol/L,37℃),且对底物腺苷和鸟苷也有磷酸解活性,在普通温度下具有较高催化活性和较好热稳定性。【结论】来源于Pseudoa lteromonas sp.XM2107的嘌呤核苷磷酸化酶在普通温度条件下具有较高的催化活性及良好热稳定性性质,在核苷类中间体和药物合成中具有较广泛的应用价值。     

枯草芽孢杆菌TM903嘌呤核苷磷酸化酶的纯化及酶学性质研究

目的：对枯草芽孢杆菌TM903嘌呤核苷磷酸化酶进行分离纯化及酶学性质研究。方法：经加热、硫酸铵盐析和SephadexG-100凝胶过滤,对枯草芽孢杆菌TM903中的嘌呤核苷磷酸化酶进行分离纯化,并对其酶学性质进行研究。结果：酶的最适反应温度为65℃,最适反应pH值为7．5,在30-50℃时热稳定性较好;K^＋对该酶有激活作用,而Na^＋、ca^＋、Mg^＋、Mn^＋等金属离子对该酶有抑制作用;Km值为2．11mmol／L,Vmax值为0．84mmol／（min·L）。结论：分离纯化了枯草芽孢杆菌TM903嘌呤核苷磷酸化酶,并研究了其酶学性质,为利巴韦林的发酵工艺优化提供了重要的酶学理论基础。     

大肠杆菌tktA基因的克隆表达

tktA是芳香族氨基酸生物合成共同途径的关键酶基因之一,在大肠杆菌中,tktA编码转酮酶A,在磷酸戊糖途径生成4－磷酸赤藓糖中起主要作用。采用PCR方法从大肠杆菌K－12株中扩增到tktA,并实现了高效表达,tktA活性提高了3．9倍,并且使芳香族氨基酸生物合成共同途径中关键中间产物DAHP的产量有所提高。     

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism.

The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2'-脱氧核苷

6-甲基嘌呤-2'-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞.本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2'-脱氧尿苷为底物合成6-甲基嘌呤-2'-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%.用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%.HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR.     

基因工程菌酶法合成抗癌新药6-甲基嘌呤-2′-脱氧核苷

6-甲基嘌呤-2′-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞。本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2′-脱氧尿苷为底物合成6-甲基嘌呤-2′-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%。用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%。HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR。     

Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus

The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.