大肠杆菌MinCD蛋白复合物纯化方法和结晶筛选

大肠杆菌在细胞分裂时,细胞中部潜在分裂位点的选择受到Min系统(Min C、Min D和Min E蛋白)的精确调控,其中Min C与Min D可以形成复合物以抑制Z分裂环在错误的位置形成。本研究采用基因克隆方法获得min C、min D基因的克隆,并表达得到Min C和Min D的重组蛋白,用亲和层析与凝胶过滤层析对所得蛋白进行纯化,通过min C、min D分别单独转化、表达纯化后混合和min C、min D共转化法两种方法得到Min CD蛋白复合物,并分别对其进行晶体筛选。结果表明,共转化法得到的Min CD复合物纯度较好,且比例约为2∶1,通过晶体筛选初步获得针状蛋白晶体,为Min CD复合物的结构解析提供了实验基础。     

大肠杆菌MinCD蛋白复合物纯化方法和晶体筛选

大肠杆菌在细胞分裂时,细胞中部潜在分裂位点的选择受到Min系统(Min C、Min D和Min E蛋白)的精确调控,其中Min C与Min D可以形成复合物以抑制Z分裂环在错误的位置形成。本研究采用基因克隆方法获得min C、min D基因的克隆,并表达得到Min C和Min D的重组蛋白,用亲和层析与凝胶过滤层析对所得蛋白进行纯化,通过min C、min D分别单独转化、表达纯化后混合和min C、min D共转化法两种方法得到Min CD蛋白复合物,并分别对其进行晶体筛选。结果表明,共转化法得到的Min CD复合物纯度较好,且比例约为2∶1,通过晶体筛选初步获得针状蛋白晶体,为Min CD复合物的结构解析提供了实验基础。     

基于MOE软件的本科生专业综合实验项目设计——耐药性金黄色葡萄球菌FtsZ抑制剂的虚拟筛选与验证

以耐药性金黄色葡萄球菌细丝温度敏感蛋白Z(FtsZ)抑制剂的虚拟筛选为例，在食品类专业综合实验课程教学中借助MOE软件，基于分子对接原理方法从花椒活性小分子库中筛选细菌FtsZ蛋白靶向抑制剂。通过配体小分子数据库的建立、受体蛋白质处理、对接参数设置、筛选结果评价和活性验证等内容，帮助学生系统掌握基于分子对接的活性分子虚拟初筛和活性验证工作流程，降低学生理解分子抑菌机制的难度，增强对跨学科交叉研究新手段的认识，拓展研究方法和创新能力。     

3MBA类FtsZ蛋白抑制剂的分子动力学模拟及抗菌作用机制

采用分子动力学模拟、蛋白质二级结构测定(DSSP)、口袋体积测量(POVME)以及MM-PBSA(molecular mechanics Poisson-Boltzmann surface area)方法, 系统研究了金黄色葡萄球菌丝状温度敏感性蛋白Z (SaFtsZ)-二磷酸鸟苷(GDP)二元复合物和SaFtsZ-GDP-3MBA (3-甲氧基苯甲酰胺)类衍生物三元复合物体系的稳定性、蛋白质二级结构、蛋白质构象、关键残基质心距、活性口袋体积以及相对结合自由能的变化规律. 研究表明: 当不含抑制剂存在时SaFtsZ-GDP二元复合物体系稳定性较差, 其T7Loop区域残基(203-209)波动较大, 且蛋白二级结构发生明显变化, 活性口袋体积急剧减小, 底物通道显著变窄且不稳定. 而含有抑制剂PC190723、Compound1 的类衍生物三元复合物体系的表现截然不同, 这主要是由于它们均能和活性口袋T7Loop区周围残基形成关键性的氢键以及疏水作用, 与FtsZ 蛋白紧密结合. 在SaFtsZ-GDP-3MBA三元复合物体系中, 3MBA仅能与活性口袋中部分残基形成疏水作用, 与FtsZ 蛋白亲和力较弱, 使其不能稳定地存在于活性口袋中, 进一步导致它的抗菌活性明显低于PC190723、Compound1. 这些发现深入揭示了3MBA类衍生物对FtsZ 蛋白的作用机制和影响规律, 为该类FtsZ 蛋白抑制剂的结构优化和产品开发应用提供了重要的理论依据.     

hBAF53 多克隆抗体的制备与活性分析

用PCR方法扩增了人的BAF53全长eDNA序列，用酶切后的△BAF53 eDNA片段构建了表达质粒．转化大肠杆菌后，经IPTG诱导表达，再经包涵体裂解、纯化，得到hBAF53抗原多肽．从免疫动物身上获得实验动物的抗血清．通过斑点印迹方法测得hBAF53抗血清的特异性和效价，又用已提取的HeLa细胞核蛋白(含有天然BAF53蛋白)进行免疫印迹分析，证明天然。BAF53蛋白是该抗血清的抗原，说明获得的抗血清具有比较高的特异性，可用于染色体改构复合物各亚基问相互作用分析及调控基因转录机理方面的研究．     

革兰氏阴性菌WaaP蛋白的表达与纯化对比研究

采用基因重组方法构建来源于大肠杆菌和铜绿假单胞菌的waa P基因的克隆,利用多种感受态细胞表达出带有不同纯化标签的可溶性Waa P蛋白,并利用亲合层析和凝胶过滤层析对可溶性Waa P蛋白进行纯化,用SDS-PAGE进行检测。对比大肠杆菌和铜绿假单胞菌中Waa P的表达和纯化结果,为蛋白结晶选取能够得到大量稳定和高纯度Waa P蛋白的表达纯化方法,并用该方法,使用硒代甲硫氨酸培养基表达出硒代甲硫氨酸标记的Waa P,为蛋白结构解析时相位的确定提供依据。     

革兰氏阴性菌WaaP蛋白的表达与纯化

采用基因重组方法构建来源于大肠杆菌和铜绿假单胞菌的waa P基因的克隆,利用多种感受态细胞表达出带有不同纯化标签的可溶性Waa P蛋白,并利用亲合层析和凝胶过滤层析对可溶性Waa P蛋白进行纯化,用SDS-PAGE进行检测。对比大肠杆菌和铜绿假单胞菌中Waa P的表达和纯化结果,为蛋白结晶选取能够得到大量稳定和高纯度Waa P蛋白的表达纯化方法,并用该方法,使用硒代甲硫氨酸培养基表达出硒代甲硫氨酸标记的Waa P,为蛋白结构解析时相位的确定提供依据。     

重组人OCIF结构域D1～D6基因在毕赤酵母中的表达

利用PCR以实验室构建的原核重组表达质粒pProEX-OCIF为模板扩增得到N末端融合有6×His标签和rTEV蛋白酶识别序列的人破骨细胞形成抑制因子(OsteoclastogenesisInhibitoryFactor,简称OCIF)结构域D1～D6(简称O CIFm)编码基因片段;将其与pMD18-T连接,转化大肠杆菌TOP10,筛选得到阳性重组质粒pMD18-OCIFm,双酶切重组克隆质粒pMD18-OCIFm得到OCIFm基因片段;将其定向插入甲醇营养型酵母分泌表达载体pPIC9中,构建获得重组表达质粒pPIC9-OCIFm.测序验证后,以限制性内切酶SalⅠ线化,电穿孔转化酵母宿主菌GS115.筛选得到阳性表达菌株后,甲醇诱导表达4d,SDS-PAGE和Westernblot对表达情况进行分析和确认.所获得的OCIFm基因片段在甲醇营养型酵母中表达量占菌体总蛋白的30%以上.利用Ni-NTA树脂对表达产物进行一步亲和层析纯化.活性测定表明纯化的表达产物可诱导体外培养的成熟破骨细胞样细胞的凋亡.表达产物的生物学活性较利用原核表达系统明显提高.     

PEI-g-MPEG/白细胞介素-10基因传递系统转染背根神经节细胞的体外性能研究

探索非病毒基因载体聚乙二醇-聚乙烯亚胺共聚物(PEI-g-MPEG)介导白细胞介素-10(Interleukin-10,IL-10)体外转染原代培养背根神经节细胞(dorsal root ganglion cells,DRGs)的效果.采用本实验室设计合成的PEI-g-MPEG,与同时携带增强型绿色荧光蛋白报告基因及IL-10基因的真核表达质粒DNA(pDC316-EGFP/IL-10)形成复合物,以脂质体(lipofectamine)复合体系Lipo/pDNA为对照,通过溴乙啶(ethidiumbromide,EB)排斥实验、凝胶阻滞电泳实验、粒径与电位的测定及扫描电镜等实验方法观察PEI-g-MPEG/pDNA的复合效果.并且检测了复合物对DRGs的毒性、转染效果及IL-10的蛋白表达情况.结果表明,PEI-g-MPEG在N/P(PEI-g-MPEG所含的氮原子和质粒DNA中磷原子的摩尔比)为5时可完全复合pDNA;随着N/P的增大,PEI-g-MPEG/pDNA复合物的粒径逐渐减小,而表面电位逐渐增大;在N/P为15时报告基因转染效果和IL-10蛋白表达情况较好,复合物的形貌呈大小均一的球形.PEI-g-MPEG/IL-10基因传递系统对于神经病理性疼痛的基因治疗具有潜在应用价值.     

来源于Alcaligenes A-6的D-氨基酰化酶基因的合成与融合表达

将来源于Alcaligenes A-6的D-氨基酰化酶基因用大肠杆菌中的丰沛密码子替换, 利用化学和基于聚合酶链反应(PCR)技术的酶促方法进行基因全合成, 利用pET-32a构建重组表达载体pET-dan, 转化进E.coil BL21(DE3)中进行融合表达. 经SDS-PAGE电泳、 Western-blot检测和活性测定发现, D-ANase可在大肠杆菌中高效表达, 目的蛋白可达到菌体总蛋白的69.2%, 密码子优化后基因构建的工程菌发酵活性为96 U/mL, 重组蛋白经超声细胞破碎及Ni²⁺柱亲和层析纯化, 比活可达1692.3 U/mg, 纯度可达95%以上.     

Targeting the bacterial Z-ring

FtsZ is a prokaryotic homolog of eukaryotic tubulin and forms the essential bacterial cell division ring (Z-ring). A new study in this issue of Chemistry & Biology, L?ppchen et al., provides further evidence that differences in nucleotide-binding properties of FtsZ and tubulin can be exploited to specifically target the bacterial Z-ring.     

Characterization and in Vitro Inhibition Studies of Bacillus anthracis FtsZ: A Potential Antibacterial Target

FtsZ is an essential bacterial cell division protein that is an attractive target for the development of antibacterial agents. FtsZ is a homologue of eukaryotic tubulin, has GTPase activity, and forms a ring-type structure to initiate cell division. In this study, the FtsZ of Bacillus anthracis was cloned into a bacterial expression vector and overexpressed into Escherichia coli BL21 (DE3) cells. The overexpressed B. anthracis FtsZ was soluble and purified to homogeneity using Ni-His-tag affinity chromatography. Like other known FtsZs, the recombinant B. anthracis FtsZ also showed GTP-dependent polymerization, which was analyzed using both spectrophotometric and Transmission Electronic Microscopic (TEM) analysis. Using the purified FtsZ, we screened a naturally extracted chemical library to identify potent and novel inhibitors. The screening yielded three chemicals, SA-011, SA-059, and SA-069, that inhibited the in vitro polymerization activity of FtsZ in the micromolar range (IC₅₀ of 55–168 μM). The inhibition potency was significantly comparable with that of berberine, a known potential inhibitor of FtsZ. Understanding the biochemical basis of the effect of these inhibitors on B. anthracis growth would provide a promising path for the development of new antianthracis drugs.     

Active membrane viscoelasticity by the bacterial FtsZ-division protein

At the early stages of the division process in Escherichia coli, the protein FtsZ forms a septal ring at the midcell. This Z-ring causes membrane constriction during bacterial division. The Z-ring associates to the lipid membrane through several membrane proteins, ZipA among them. Here, a simplified FtsZ-ZipA model was reconstituted onto Langmuir monolayers based in E. coli polar lipid extract. Brewster angle and atomic force microscopy have revealed membrane FtsZ-polymerization upon GTP hydrolysis. The compression viscoelasticity of these monolayers has been also investigated. The presence of protein induced softening and fluidization with respect to the bare lipid membrane. An active mechanism, based on the internal forces stressed by FtsZ filaments and transduced to the lipid membrane by ZipA, was suggested to underlie the observed behavior.     

Probing FtsZ and tubulin with C8-substituted GTP analogs reveals differences in their nucleotide binding sites

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.     

Mechanism of Action of the Cell-Division Inhibitor PC190723: Modulation of FtsZ Assembly Cooperativity

The cooperative assembly of FtsZ, the prokaryotic homologue of tubulin, plays an essential role in cell division. FtsZ is a potential drug target, as illustrated by the small-molecule cell-cycle inhibitor and antibacterial agent PC190723 that targets FtsZ. We demonstrate that PC190723 negatively modulates Staphylococcus aureus FtsZ polymerization cooperativity as reflected in polymerization at lower concentrations without a defined critical concentration. The crystal structure of the S. aureus FtsZ-PC190723 complex shows a domain movement that would stabilize the FtsZ protofilament over the monomeric state, with the conformational change mediated from the GTP-binding site to the C-terminal domain via helix 7. Together, the results reveal the molecular mechanism of FtsZ modulation by PC190723 and a conformational switch to the high-affinity state that enables polymer assembly.     

Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets

The Min proteins from E.coli position the bacterial cell‐division machinery through pole‐to‐pole oscillations. In vitro, Min protein self‐organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane‐enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell‐division protein FtsZ‐mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ‐mts structures on compartment size. Finally, co‐reconstitution of Min proteins and FtsZ‐mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self‐organization in confined volumes.     

Interaction of Cu-dipeptide complexes with Calf Thymus DNA and antiproliferative activity of [Cu(ala-phe)] in osteosarcoma-derived cells

In this work the study of Calf Thymus DNA interaction with several Cu(l-dipeptide) complexes was reported. The binding stoichiometry (Cu(mmol)/DNAmol base) was determined and in an attempt to clarify the binding mode, EPR and CD experiments were performed. All the studied complexes interacted with DNA, in a more selective way than [Cu(H₂O)₆]²⁺, being the [Cu(ala-phe)] the complex with the highest interaction. The EPR experiments suggested that the monomeric species formed in solution were coordinated through a nitrogen atom of the DNA bases (inner-sphere binding) and the CD studies showed structural changes upon the DNA–complex interaction. Besides, the ratio Cu(mmol)/DNAmol base obtained by the binding stoichiometry experiments was close to that found by EPR and CD determinations. The effect on cell proliferation determined by the crystal violet bioassay on UMR106 rat osteosarcoma-derived cells showed that the [Cu(ala-phe)] complex exerted an antiproliferative action against this tumor line.     

3种色谱模式联用在中药活性成分初步筛选中的应用

提出了反相高效液相色谱(RP-HPLC)、固定化脂质体色谱(ILC)以及固定化载体蛋白色谱(ICPC)3种色谱模式联用筛选中药中活性成分的新思路，并用于传统中药川芎中的生物活性成分的初步筛选。从川芎的甲醇提取液中筛选出几种既有细胞膜的穿透能力又有与载体蛋白的结合能力的成分，并对其中两种主要的组分进行了初步的结构鉴定。     

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.     

Recombinant Expression and Characterization of l-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5?°C, was isolated from cow dung; l-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37?°C and in a buffer system of pH?6 to 7. The enzyme exhibited thermostability at 50?°C with a 33% and 28% of activity retention after 45 and 60?min. The kinetic parameters for the enzyme were calculated from Lineweaver?CBurk plot, and K _m and V _max were 0.89?mM and 0.18?U/mg, respectively.