hHO-1结构预测及突变体的构建、表达、纯化和活性测

人血红素加氧酶-1(human heme oxygenase-1,hHO-1)是血红素代谢的限速酶,直接调节体内胆红素水平.利用软件Spdbv程序对hHO-1进行结构模拟预测,以Ala取代第25位His残基,hHO-1活性部位的结构有较明显的改变,但据模拟推测突变后hHO-1与血红素仍然具有结合性.依据模拟结果,构建野生型和突变体表达载体pBHO-1和pBHO-1(M),并分别转化大肠杆菌DH5α,IPTG诱导表达目的蛋白.表达产物经30%-60%(NH4)2SO4盐析后纯度提高3.6倍,再经两次Q-Sepharose Fast Flow阴离子交换树脂分离则表达产物的纯度提高30倍.酶活性测定显示,突变体hHO-1(△hHO-1)较野生型hHO-1(whHO-1)活性下降了91.21%.本研究显示hHO-1的第25位His在酶与底物血红素氧化反应中起着重要作用,为有效调控酶活性发挥其生物学作用提供依据.     

人血红素加氧酶-1的原核表达、纯化及其中和抗体的制备

目的确定人血红素加氧酶-1（human heme oxygenase-1,hHO-1）在大肠埃希菌中的表达条件与纯化方法,利用纯化的蛋白制备具有中和活性的hHO—1多克隆抗体。方法将hHO-1原核表达质粒pMW172／hHO-1转人大肠埃希菌菌株BL21,通过改变摇床转速、诱导剂IPTG浓度和培养时间确定hHO-1蛋白的最佳可溶性表达条件;利用超声破碎、高速离心、分级盐析、分子筛层析等方法纯化hHO-1蛋白,建立体外HO-1活性测定方法检测hHO-1蛋白的活性;利用纯化的hHO-1活性蛋白作为抗原免疫新西兰兔,制备多克隆抗体;利用ELISA方法和Western印迹技术分别测定抗体的效价和特异性,通过HO-1活性测定检测抗体的中和活性。结果确定hHO-1最佳可溶性表达条件为：37℃、200r／min培养3h后,0．1 mmoL／LIPTG诱导培养4h。超声破碎菌体,上清经30％～40％盐析纯化及分子筛层析纯化,获得活性hHO-1蛋白,收得率为30．3％,纯化倍数为2．83倍,纯度为90％。制备的抗hHO-1的兔血清效价达到10^6,并能中和掉46％hHO-1的催化活性。结论为hHO-1蛋白的表达和纯化以及多克隆抗体制备确立了可行的技术方案;获得了高纯度活性hHO-1蛋白及hHO-1多克隆抗体,为HO-1功能、结构研究,以及相关疾病研究奠定了基础。     

用siRNA沉默人血红素加氧酶-1基因降低胆红素水平

拟通过RNA干扰技术特异下调人血红素加氧酶-1(human heme oxygenase-1,hHO-1)基因的表达,减少hHO-1的产量从而降低胆红素的产生,探讨在胆红素产生前就阻断其产生,为临床早期防治新生儿高胆红素血症及胆红素中毒性脑病探索一种新的有效手段。针对hHO-1基因设计并化学合成三对小分子干扰RNA(small interfering RNA,siRNA)。采用脂质体转染法将siRNA转染入人肝脏细胞株HL-7702;荧光显微镜检测siRNA转染细胞的效率;转染siRNA1~2天后经RT-PCR和Western印迹方法检测hHO-1表达水平和蛋白质量;并采用HO-1诱导剂血红素诱导或hHO-1表达质粒转染细胞以上调hHO-1表达,检测siRNA干扰后hHO-1产量和酶活性。结果显示:设计的三对siRNA能不同程度的特异下调hHO-1表达,筛选获得抑制效果最佳的siRNA-3。siRNA-3抑制hHO-1呈现浓度与时间依赖性。与非特异对照siRNA及未处理组比较,血红素诱导和hHO-1表达质粒转染均能上调HL-7702细胞内hHO-1表达,提高hHO-1产量,但转染siRNA-3后hHO-1表达明显抑制,同时hHO-1活性随着基因表达下调而下降。实验表明设计合成的siRNA-3抑制效果明显。siRNA-3通过降解hHO-1,减少hHO-1产量,降低酶活性,最终减少胆红素产生,从而使RNA干扰技术成为降低新生儿高胆红素血症和胆红素中毒性脑病发生的一种候选方法。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

枯草蛋白酶E的突变体

Ser236位于横贯枯草蛋白酶E的α螺旋末端,远离催化活性中心,Ser236的突变不会对酶的活性产生大的影响。用定点突变的方法对枯草蛋白酶E的基因进行改造引入Ser236Cys,可能会形成分子间二硫键,有利于提高酶的稳定性。Ser236Cys变体酶(BP1)活性是野生型蛋白酶E的15倍,热稳定性提高3倍;进一步在其他位点引入突变的变体酶BU1(A1a15Asp/Gly20His/Ser236Cys)和BW1(Ser24His/Lys27Asp/Ser236Cys)活性都比野生型蛋白酶E低,但BW1的稳定性稍高于野生型蛋白酶E。     

转染人血红素加氧酶-1基因对大鼠血管平滑肌细胞抗氧化损伤的作用

本实验构建含人血红素加氧酶-1（hHO-1)基因的逆转录病毒载体XM-6/hHO-1,将其导入离体培养的大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC),观察外源性hHO-1基因在VSMC内的表达及其抗活性氧损伤作用,结果表明：（1）hHO-1基因可在靶细胞中明显表达,转染VSMC的HO-1蛋白表达和HO酶活性分别比非转染细胞高1.8倍和2.0倍;（2）转染hHO-1的VSMC可对抗大剂量H2O2对细胞的损伤作用,表现为细胞存活率增加和乳酸脱氢酶（LDH）漏出减少,上述保护作用可被HO特异性抑制剂锌原卟啉IX（Zinc-proto-porphyrinIX,ZnPP-IX)所阻断,研究结果提示,外源性HO-1的过量表达可增加VSMC对抗氧化损伤的能力。     

嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。     

人碱性成纤维细胞生长因子突变体的高效表达

用PCR法将人碱性成纤维细胞生长因子(hbFGF)基因中编码第25、69和92位的半胱氨酸(Cys)密码子突变为丝氨酸(Ser)密码子,将突变的hbFGFcDNA片断与表达质粒pET3c连接,构建重组质粒pET3chbFGFSer25,69,92。hbFGFSer25,69,92在大肠杆菌BL21(DE3)中的表达量大于30%。通过阳离子交换和肝素亲和层析两步纯化,得到纯度大于95%的hbFGFSer25,69,92。MTT法测定纯化的产物活性表明,hbFGFSer25,69,92突变体促Balb/c细胞增殖的活性与野生型hbFGF相当,为下一步对hbFGFSer25,69,92突变体进行定点化学修饰打下了基础。     

基于易错PCR的假密环菌Armillariella tabescens MAN47β-甘露聚糖酶耐高温定向进化

本研究利用易错PCR技术突变假密环菌Armillariella tabescens MAN47β-甘露聚糖酶野生型基因,PCR产物与大肠杆菌-酿酒酵母穿梭表达载体pYCα上连接,在大肠杆菌DH5α中扩增后电转入酿酒酵母Saccharomyces cerevisiae,构建了库容为10_4的初级突变体库,筛选得到耐高温最佳突变株M262.DNS法测得80℃处理30 min后最大酶活力为25 U/mL,较之野生型最适条件酶活力提高了4.3倍.序列分析表明,突变体有3个碱基发生了突变:T343A/C827T/T1139C,相应的氨基酸改变为Ser115Thr/Thr276Met/Val380Ala,利用SWISS-MODEL数据库同源建模显示,这3个突变氨基酸分别位于第4个13折叠的第6个氨基酸、第6个α螺旋的第1个氨基酸、第10个α螺旋和第11个β折叠之间的转角.     

Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand

To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman’s reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.     

Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase

Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.     

Isolation and Purification of Cytokinin-Binding Protein from Wheat Chloroplasts

The extract of wheat chloroplast membrane proteins was precipitated by different saturation of (NH4)2SO4. Pellet of 0–30% saturation showing high binding activity to 3H-6BA wax loaded on the affinity chromatography column which was prepared by coupling 6BA to epoxy activated sepharose 6B. The CTK-binding protein was eluted from the BA-sepharose 6B column with Tris buffer containing 0.1 mmol/L 6BA. It showed a single protein band on PAGE and the apparent molecular weight was about 250kD. Two bands with molecular weight of 60kD and 66kD were detected on SDS-PAGE. It was supposed that the protomer of CTK-binding protein was a tetramer of two subunits.     

A Proteinase from Mung Bean Sprouts That Inactivaties Soybean Trypsin Inhibitor (in English)

By 30%-60% (NH4)2SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (ProteinPM-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean (Vigna rabiata (L.) Wilczek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its Km and Vmax for STI were 769.2N-α-benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE·mL-1·min-1 respectively. This proteinase was stable at temperatures lower than 50℃ and pH 6.5-8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50℃ and pH 8.0 in 4 h.     

大鼠骨骼肌多催化功能蛋白酶的提取、鉴定及其抗血清的制备

为了研究多催化功能蛋白酶（ｍｕｌｔｉｃａｔａｌｙｔｉｃａｌｐｒｏｔｅｉｎａｓｅ,ＭＣＰ）在负氮平衡形成中的作用,以大鼠骨骼肌为原料,提取此酶并制备其抗血清．将大鼠骨骼肌粗提物经４５％～６５％饱和度硫酸铵分级盐析、阴离子交换层析和凝胶过滤,最后从Ｓｅｐｈａｒｏｓｅ４Ｂ层析柱上获得单一活性洗脱峰．酶活性用Ｃａｒｂｏｂｅｎｚｏｘｙ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－４－ｎｉｔｒｉｎｉｌｉｄｅａｃｅｔａｔｅ作底物检测．非变性ＰＡＧＥ银盐染色显示单一区带的骨骼肌多催化功能蛋白酶,ＳＤＳ－ＰＡＧＥ银盐染色显示１０条亚基电泳区带,分子量在２５～３２ｋＤ之间．纯化的酶免疫兔８周后,抗血清效价达１３２,用分级盐析和离子交换层析纯化抗血清,显示单一电泳区带的ＩｇＧ．Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析显示只在２５～３２ｋＤ之间出现多条亚基区带．这些结果提示已获得电泳纯ＭＣＰ及其较高特异性的多克隆抗体．     

苦瓜籽核糖体失活蛋白的理化性质及生物活性

采用硫酸铵分级分离,假配基亲和层析和ＳｅｐｈａｃｒｙｌＳ－１００分子筛层析等方法,从苦瓜籽中获得核糖体失活蛋白（ＲＩＰ）．经ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ、ＩＥＦ和ＰＡＳ方法分析均表明为单一蛋白着色带或单一糖蛋白着色带．根据ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ Ｇ－１５０分子筛层析结果计算其相对分子质量为３．０×１０４,经ＩＥＦ－ＰＡＧＥ结果计算其ｐＩ为８．９～９．０．对无细胞系统中蛋白质生物合成抑制活性明显,其ＩＣ５０为５．３×１０－ １０ ｍ ｏｌ／Ｌ左右．体外生物活性试验结果表明其对人肝癌细胞、Ｖｅｒｏ、ＳＰ２／０、３Ｔ３、Ｋｂ、Ｎａｖａｎａ 等肿瘤细胞株均表现有不同程度的抑制作用．而对完整细胞人胚肺二倍体细胞却毒性极小．因此,上述实验结果为该ＲＩＰ的进一步深入研究和有可能开发成免疫毒素的高效弹头药物提供了一定的工作基础．     

粘质赛氏菌胞外蛋白酶的提取、纯化及部分性质研究

经过硫酸铵３０％～５０％分级沉淀、二步柱层析可获聚丙烯酰胺凝胶电泳均一的粘质赛氏菌胞外蛋白酶制品,收率可达５３％,并制备了酶的结晶,该酶以ＳｅｐｈａｄｅｘＧ１００柱层析及ＳＤＳ－ＰＡＧＥ测得分子量约为８１０００,该酶的最适ｐＨ为７．０,最适温度为４５℃,Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋、Ｃｕ２＋、Ｃｏ２＋等重金属离子不同程度地抑制酶活性。     

Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.     

Hydroxymethylglutaryl-coenzyme A reductase. Purification and properties of the enzyme from Fusarium oxysporum.

The hydroxymethylglutaryl-coenzyme A reductase (mevalonate:NADP+ oxidoreductase, EC 1.1.1.34) system in Fusarium oxysporum, a soil inhabiting plant pathogen, has been examined. Two forms of the enzyme catalyzing the conversion of hydroxymethylglutaryl-coenzyme A were obtained in the supernatant after precipitation at 75% (NH4)2SO4 saturation of the soluble culture extract which was previously separated from cell wall, mitochondria and microsomes. The two forms of the enzyme were separated electrophoretically. A third form, contained in the precipitate obtained at 35--75% (NH4)2SO4 saturation of the same extract, was further purified by Sephadex G-50 column chromatography. This purified form moved as a single band in sodium dodecyl sulphate electrophoresis and in immunological tests and has a molecular weight of 11 000. The apparent Michaelis constant for the substrate hydroxymethylglutaryl-coenzyme A is 21 micron at 2 micron NADP. NADPH is a more efficient reductant on a molar basis than NADH for the deacylation of the hydroxymethylglutaryl-coenzyme A substrate. Optimum activity of the enzyme was obtained at pH 7.4 and 37 degrees C. The enzyme demonstrated no cold sensitivity but rather was more stable at 4 degrees C than at 25 degrees C. The protection with dithiothreitol, though minimal compared to other systems, was more effective at the higher temperature.