Name: one-step negative chromatographic purification of helicobacter pylori neutrophil-activating protein overexpressed in escherichia coli in batch mode
Uploaded: 2016-06-18T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode at JoVE.co

We thank Dr. Chao-Sheng Cheng at National Tsing Hua University, Taiwan, for performing the circular dichroism measurement. We also thank Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA, for providing the anti-HP-NAP monoclonal antibody. We appreciate Drs. Han-Wen Chang and Chung-Chu Chen at Mackay Memorial Hospital, Hsinchu, Taiwan, for providing advice for IRB application, Mr. Te-Lung Tsai at the Mackay Memorial Hospital, Hsinchu, Taiwan, for supervising the analysis of isolated neutrophils, and Ms. Ju-Chen Weng at National Tsing Hua University, Taiwan, for her technical assistance. This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 104-2311-B-007-003, NSC101-2311-B-007-007 and NSC98-2311-B-007-006-MY3), the Joint Research Program of National Tsing Hua University and Mackay Memorial Hospital (100N7727E1, 101N2727E1, 103N2773E1), and the research program of National Tsing Hua University (104N2052E1).

人体的血液是由来自清大的机构审查委员会，台湾新竹书面知情同意和批准健康志愿者收集。 1. E.重组HP-NAP的表达大肠杆菌 <ol><li>准备含H. HP-NAP的DNA序列的质粒pET42a-NAP 幽门螺杆菌 26695菌株如前所述16。制备含有HP-NAP与如所描述的所需的点突变的DNA序列的质粒（见协议，步骤8）。 </li><li>变换上述DNA质粒导入大肠杆菌的10毫微克大肠杆菌 BL21（DE3）感受态细胞通过45秒钟的热休克在42℃下，条纹的琼脂含有50μg/ ml卡那霉素，和在37℃下孵育板16小时板上的细胞上LB培养基（LB）。 </li><li>接种在含有5毫升LB培养液的单独的管单菌落用50微克/ ml卡那霉素，并在3成长它们作为预培养7℃，用在170 rpm振摇16小时。 </li><li>接种将2ml上述预培养的细胞在200ml LB肉汤中含有50μg/ ml卡那霉素的1升烧瓶中。孵育在37℃下接种培养瓶2小时以在170 rpm振摇直到在600nm的吸光度达到约用UV / VIS分光光度计检测0.4-0.5。 </li><li>添加80微升的1M异丙基β-D-1-硫代半乳糖苷（IPTG）的入上述培养到0.4毫米的最终浓度来诱导的HP-NAP的表达。孵育3小时的培养，直到在600nm的吸光度达到约1.6-1.7。 </li><li>离心15分钟将细胞以6000 xg离心在4℃下，以除去上清液。在-70℃下直到纯化存储细胞沉淀。 </li></ol> 2.可溶性的蛋白质部分含有HP-NAP的制备注意:所有以下步骤在4℃进行。 <ol><li>重悬将50ml单元p的在协议步骤1.6中ellet制备20毫升的20mM的Tris-HCl，pH值9.0，50mM NaCl中含有0.13毫苯甲磺酰氟（PMSF），0.03毫摩尔N-二的α-甲苯磺酰基-L-赖氨酰 - 氯甲基酮（TLCK），和0.03的毫N-甲苯磺酰基-L- phenylalaninyl-氯甲基酮（TPCK）在4℃下，如前所述16。 </li><li>通过在4℃下通过细菌悬浮液通过在范围为15,000至20,000磅操作7次高压均质破坏细菌细胞。 </li><li>离心机在30,000×g下细胞裂解物在4℃下1小时，通过使用超速离心分离可溶和不溶的蛋白组分。转移上清液作为可溶性蛋白质部分到烧杯中。 </li><li>通过使用用牛血清白蛋白（BSA）的商业试剂盒为标准，根据制造商的说明Bradford方法测定可溶性蛋白级分的蛋白浓度。 </li><li>添加177.6微升的1N HCl于20ml日。从协议步骤2.3获得的在pH 9.0将其pH值调节至pH 8.0È可溶性蛋白质部分。 </li><li>添加20毫摩尔的Tris-HCl，pH值8.0，50mM NaCl中，以上述蛋白质溶液，使最终蛋白质浓度为0.5毫克/毫升。 </li></ol> 3.重组HP-NAP从大肠杆菌纯化大肠杆菌通过用DEAE离子交换树脂负态批处理色谱<ol><li>准备15毫升DEAE树脂。 <ol><li>权衡的DEAE树脂的出0.6克干粉，并暂停其在30毫升的20mM的Tris-HCl，pH值8.0，50mM NaCl中的在室温下至少1天。 </li><li>离心树脂10,000× 下于4℃下1分钟。 </li><li>取出并弃去上清液。 </li><li>添加20毫摩尔的Tris-HCl，pH值8.0，50mM NaCl中15毫升</li><li>重复步骤协议3.1.2 3.1.4四次。 </li><li>存储15毫升4℃用于后续结算树脂（在30ml Tris缓冲液的50％浆料）。 注意:所有下列步骤的s的在4℃下进行。 </li></ol></li><li>添加45毫升在协议步骤2.6制得的可溶性蛋白的〜15的树脂的溶液，并搅拌以在4℃下磁力搅拌1小时蛋白质/树脂浆。 </li><li>倾蛋白质/树脂浆成装配有旋塞的塑料或玻璃柱。使树脂在重力作用下沉降。 </li><li>打开活塞，使蛋白质溶液通过重力流柱运行，直到在塔中的液面刚好树脂上方。收集流通作为未结合的级分，其中包含纯化的HP-NAP。 </li><li>加入15mL冰冷的20mM的Tris-HCl，pH值8.0，50mM NaCl中的入列。 </li><li>打开活塞，以允许洗涤缓冲液，以通过重力流动柱运行，直到在塔中的液面刚好树脂上方。收集流通作为洗涤级分。 </li><li>重复步骤协议3.5〜3.6四次收取额外的洗涤流份。 </li><li>加入15mL冰冷的20mM的Tris-HCl，pH值8.0，1M NaCl的成一列。 </li><li>打开活塞，使洗提缓冲液向通过重力流动柱运行，直到在塔中的液面刚好树脂上方。收集流通作为洗脱级分。 </li><li>重复步骤协议3.8到3.9四次收取额外的洗脱份。 </li></ol> 4.缓冲液交换，并通过与DEAE树脂负片模式批量层析纯化HP-NAP的内毒素清除注:HP纯化-NAP在大肠杆菌中表达大肠杆菌需要进行缓冲液交换，并刺激嗜中性粒细胞之前去除内毒素。 <ol><li>执行透析用透析管以截留分子量14千道尔顿如前所述24至纯化的HP-NAP的缓冲器改变为Dulbecco氏磷酸盐缓冲盐水（D-PBS），pH值7.2，在4℃下。 </li><li>通过syring过滤透析HP-NAP与流速在4℃下范围从2.5至4毫升/分钟，以除去内毒素附着到20ml的一次性注射器带正电的，亲水膜ê滤波器。 </li></ol> 5.表征十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），Western印迹，本地-PAGE，凝胶过滤层析，和圆二色光谱纯化的重组HP-NAP的分子性质<ol><li>分析通过SDS-PAGE和免疫印迹用含有小鼠单克隆抗体单克隆16F4 25杂交瘤培养上清中纯化重组的HP-NAP如前所述24。 </li><li>分析由天然-PAGE和凝胶过滤色谱法的纯化，HP-NAP如前所述26以检查其寡聚状态。 </li><li>通过圆二色光谱分析纯化的重组的HP-NAP如前所述26以检查其二级结构。 </li></ol> 6. HP-NAP的通过SDS-PAGE胶的银染纯度评价<ol><li>根据银染色试剂盒的制造商的说明制备定影液，敏化溶液，银溶液，显影溶液，停止溶液和洗涤液。 </li><li>根据银染色试剂盒的制造商的说明进行的SDS-PAGE凝胶的银染色。 </li><li>获得凝胶的图像与成像系统。 </li></ol> 7. ROS产生的测量中性粒细胞通过HP-NAP诱导<ol><li>如前所述24通过葡聚糖沉降，随后通过密度梯度离心分离从人肝素化血液的人类嗜中性粒细胞。 </li><li>中性粒细胞ROS产生的测量用鲁米诺依赖的化学发光法与HP-NAP刺激。 <ol><li>打开读板器，并设置在37℃的温度。放置一个空平底96孔白色板读板器腔室内部，使其温热至37℃。 </li><li>制备刺激混合物在D-PBS中，pH值7.2，含0.9毫氯化钙 ，0.5mM的MgCl 2的，和13.3μM5-氨基-2,3-二氢-1,4-酞嗪二酮（鲁米诺）与存在和不存在从协议的步骤准备4.2μM0.67毒素去除HP-NAP。 </li><li>调整从协议步骤制备的D-PBS，pH 7.2的7.1至2×10 6个细胞/ ml，含有5mM葡萄糖的人中性粒细胞的浓度。 </li><li>加入50μl嗜中性粒细胞悬浮液到96孔而板的各孔中。 </li><li>加入150微升从协议的步骤7.2.2制备成井含有嗜中性粒细胞在每孔5秒的时间间隔的刺激混合物。 </li><li>通过使用平板读数器测量化学发光的为每孔5秒的时间内三小时发射。 </li></ol></li></ol> 8。聚合酶链反应（PCR）的DNA质粒窝藏HP-NAP突变体的建设为基础的站点直接诱变注意:作为除了"沉默"限制性位点通过定点诱变（SDM） -协助软件28引入到诱变引先前27所述的基本生成基于PCR的位点直接诱变。 <ol><li>进行PCR反应。 <ol><li>加入10纳克质粒pET42a-NAP的， 表1中所列的诱变引物对的1微米，脱氧核苷三磷酸（dNTPs浓度），和3个单位的高保真PCR酶混合物的成PCR管含有去离子水的200微米，得到最终的25微升体积。 </li><li>启动PCR循环在95 ℃下进行10分钟以变性模板DNA，并遵循具有12个扩增循环在95℃1分钟，Tm 值没有 -5℃1分钟，6分钟72 摄氏度 。 </li><li>完成PCR循环用在的Tm第 -5℃下的退火步骤1分钟，在72℃的延伸步骤为30分钟。 </li></ol></li><li>治疗15微升的PCR产物用0.4微升DPN限制性酶于37℃2小时，然后用琼脂糖凝胶电泳分析2微升DPN我处理的PCR产物。 </li><li>屏幕上的突变体。 <ol><li>变换2微升PCR产物到大肠杆菌大肠杆菌 DH5α感受态细胞的热休克45秒，在42℃。 </li><li>涂布在含有50μg/ ml卡那霉素的LB平板转化细胞，并在37℃下孵育板16小时。 </li><li>隔离从细菌菌落使用根据制造商的方案在商业碱性裂解试剂盒的质粒DNA。 </li><li>治疗在协议步骤8.3.3用XhoI限制性酶分离，以验证根据制造商的协议所需的沉默突变的存在的质粒DNA。 </li><li>序列质粒DNA核实协议步骤8.3.4用T7启动子引物，以确认根据制造商的协议的HP-NAP突变体的编码序列的校正。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+ion-exchange+column+chromatography+of+proteins:+Comparison+of+different+formats.">Large-scale ion-exchange column chromatography of proteins: Comparison of different formats.</a> J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 790 (1-2), 17-33 (2003).</li><li>Lee, M. F. X., Chan, E. S., Tey, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+chromatography:+Progress,+applications+and+future+perspectives.">Negative chromatography: Progress, applications and future perspectives.</a> Process Biochem. 49 (6), 1005-1011 (2014).</li><li>Yang, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+yield+purification+of+Helicobacter+pylori+neutrophil-activating+protein+overexpressed+in+Escherichia+coli.">High yield purification of Helicobacter pylori neutrophil-activating protein overexpressed in Escherichia coli.</a> BMC biotechnol. 15 (23), (2015).</li><li>Iankov, I. D., Haralambieva, I. H., Galanis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+attenuated+measles+virus+engineered+to+express+Helicobacter+pylori+neutrophil-activating+protein.">Immunogenicity of attenuated measles virus engineered to express Helicobacter pylori neutrophil-activating protein.</a> Vaccine. 29 (8), 1710-1720 (2011).</li><li>Shih, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+chromatographic+purification+of+Helicobacter+pylori+protein+expressed+in+Bacillus+subtilis.">One-step chromatographic purification of Helicobacter pylori protein expressed in Bacillus subtilis.</a> PLoS One. 8 (4), e60786 (2013).</li><li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC biotechnol. 8 (91), (2008).</li><li>Karnik, A., Karnik, R., Grefen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDM-assist+software+to+design+site-directed+mutagenesis+primers+introducing+'silent'+restriction+sites.">SDM-assist software to design site-directed mutagenesis primers introducing 'silent' restriction sites.</a> BMC bioinformatics. 14 (105), (2013).</li></ol>

HWF and YCY are inventors of patents TW I 432579 and US 8,673,312 for the method of one-step purification of Helicobacter pylori neutrophil-activating protein. All materials described in the manuscript will be available for research purposes. The authors confirm that this does not alter their adherence to all of the JoVE's policies on sharing data and materials.

负模式批处理色谱用DEAE阴离子交换树脂这里介绍的是适用于在大肠杆菌中过表达重组的HP-NAP的纯化。在细胞裂解和纯化的步骤中使用的缓冲液的pH值是非常关键的，以确保的HP-NAP的在大肠杆菌中的溶解度大肠杆菌裂解液和重组HP-NAP从宿主细胞的杂质，分别为高效分离。细菌细胞应在pH9.0被裂解，和负纯化应在pH8.0下进行，以获得高产率和高纯度的HP-NAP。 HP-NAP的典型的恢复率为90...

幽门螺杆菌 （ 幽门螺旋杆菌 ）是胃炎和消化性溃疡的主要诱因。这种细菌也被列为被国际癌症研究机构，世界卫生组织的一部分，人类致癌物，在1994年据估计， 幽门的流行幽门螺杆菌感染是在发展中国家70％，在工业化国家1 30-40％。即使H的感染率幽门螺旋杆菌是减少在工业化国家，H.感染率幽门螺杆菌在发展中国家仍高2。标准治疗根除H.幽门螺杆菌感染是由质子泵抑制剂，PPI和两种抗生素，克拉霉素加阿莫西林或甲硝唑3的管理。然而，在H·抗生素抗性的上升幽门 -相关溃疡治疗敦促新的战略，以防止澳发展- [R治愈感染。针对H.预防和/或治疗性疫苗接种的发展幽门螺杆菌可以提供另一种方法来控制H.幽门螺杆菌感染。 幽门螺杆菌中性粒细胞激活蛋白（HP-NAP）， 幽门螺旋杆菌的主要致病因子，在H的水萃取物最初被鉴定幽门螺杆菌与激活嗜中性粒细胞粘附到内皮细胞和产生活性氧（ROS）4的能力。胃黏膜中性粒细胞浸润在H.发现幽门螺旋菌感染的患者活动性胃炎，可能会导致在胃中的炎症和组织损伤。因此，HP-NAP可以通过激活的中性粒细胞发挥 ​​病理作用以诱导胃炎，这进一步导致溃疡或H.幽门螺杆菌机相关胃部疾病。然而，HP-NAP是临床应用5,6的潜在候选。由于免疫原性和免疫调节亲HP-NAP的perties，这种蛋白可用于开发疫苗，治疗剂，和诊断工具。临床试验已经对利用重组的HP-NAP已经进行作为对H的蛋白疫苗的成分之一幽门螺旋杆菌 。此疫苗由重组的HP-NAP，细胞毒素相关基因的（相关蛋白），并用氢氧化铝配制空泡毒素A（毒素）的蛋白质，并已进一步证明是安全和人类中7的免疫原性。此外，HP-NAP充当一个强有力的免疫调节剂，以引发T辅助1型（Th1细胞）偏振光的免疫应答用于癌症治疗8和下调的过敏反应和寄生虫感染9,10-诱发Th2细胞介导的免疫反应。作为用于诊断的，重组的HP-NAP-ELISA已经被应用到检测针对H. HP-NAP血清抗体幽门螺旋杆菌感染的患者11。一项研究表明，HP-NAP特异性抗体的血清水平从H. PY洛瑞感染的胃癌患者较显著高于从慢性胃炎患者12。另一项研究也显示，依靠HP-NAP血清抗体与非贲门胃腺癌13的存在相关联。因此，重组的HP-NAP-ELISA可应用于检测抗HP-NAP血清抗体为H.胃癌预后幽门螺旋杆菌感染的患者。两者合计，纯化的HP-NAP可进一步用于预防，治疗，及H的预后幽门机相关的疾病以及癌症的治疗。 在用于重组的HP-NAP的纯化的几种方法中的天然形式的大肠杆菌 （ 大肠杆菌 ）表达报道，到目前为止，还需要一第二纯化步骤涉及凝胶过滤层析，得到高纯度的HP-NAP 14-16 。这里，使用负模式间歇色谱法的方法二乙基氨基乙基（DEAE） -离子交换树脂是针对在大肠杆菌中过表达的HP-NAP的纯化描述大肠杆菌高产和高纯度。该纯化技术是基于结合宿主细胞蛋白质和/或比的HP-NAP至树脂等杂质。在pH 8.0，除HP-NAP几乎没有其他的蛋白质与未结合的级分回收。采用DEAE离子交换色谱法在负模式该纯化方法是简单的和时间通过允许经由一步法色谱重组的HP-NAP的纯化通过未结合部分的集合保存。除了​​HP-NAP，其他几个生物分子，如病毒17，免疫球蛋白G（IgG）的18，血红蛋白19，蛋白磷酸酶20，和毒力因子鞭毛蛋白21，也有报道可以通过离子交换层析在负纯化模式。负模式是优选的离子交换色谱法，如果杂质是次要本进行样品中组分待纯化22。在天然或重组生物分子净化负面色谱的应用程序已被最近检讨23。 本报告提供了在大肠杆菌重组HP-NAP表达一步步协议大肠杆菌 ，细胞的溶胞，并且使用负模式间歇色谱用DEAE离子交换树脂HP-NAP的纯化。如果需要纯化的蛋白是适合于在负模式离子交换层析，所描述的协议也可以适于作为起点的纯化过程的发展。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET42a-NAP&amp;nbsp;</td><td>N/A</td><td>N/A</td><td>prepared as described in Supplementary data of Refernce 15&amp;nbsp;&lt;br/&gt; http://www.sciencedirect.com/science/article/pii/S0006291X08018317</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 (DE3)</td><td>Thermo Fisher Scientific Inc</td><td>C6000-03</td><td>https://www.thermofisher.com/order/catalog/product/C600003</td></tr><tr><td>Kanamycin</td><td>Amresco</td><td>25389-94-0</td><td>http://www.amresco-inc.com/KANAMYCIN-SULFATE-0408.cmsx</td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>MD Biomedical Inc</td><td>101-367-93-1</td><td>http://www.antibody-antibodies.com/product_det.php?id=238064&amp;amp;supplier=search&amp;amp;name&lt;br/&gt;=IPTG%20</td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma-Aldrich</td><td>10837091001</td><td>protease inhibitor&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/roche/PMSFRO?lang=en&amp;amp;region=TW</td></tr><tr><td>N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK)</td><td>Sigma-Aldrich</td><td>T7254</td><td>protease inhibitor&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/t7254?lang=en&amp;amp;region=TW</td></tr><tr><td>N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK)</td><td>Sigma-Aldrich</td><td>T4376</td><td>protease inhibitor&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/t4376?lang=en&amp;amp;region=TW</td></tr><tr><td>Protein standard (bovine serum albumin)</td><td>Sigma-Aldrich</td><td>P5619&amp;nbsp;</td><td>a standard protein for Bio-Rad Protein Assay&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/fluka/p5619?lang=en&amp;amp;region=TW</td></tr><tr><td>DEAE&amp;ndash;Sephadex &amp;nbsp;A-25 chloride form</td><td>Sigma-Aldrich</td><td>A25120</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/a25120?lang=en&amp;amp;region=TW</td></tr><tr><td>Spectrum/Por dialysis tubing</td><td>Spectrum Laboratories</td><td>132720</td><td>with molecular weight cutoff of 14 kDa&lt;br/&gt; http://www.spectrumlabs.com/dialysis/RCtubing.html?Pn=132720;</td></tr><tr><td>Acrodisc&amp;reg; units with Mustang&amp;reg; E membrane</td><td>Pall</td><td>MSTG25E3</td><td>for endotoxin removal; operated at flow rates ranging from 1 to 4 ml/min&lt;br/&gt; http://www.pall.com/main/laboratory/product.page?id=19992</td></tr><tr><td>mouse monoclonal antibody MAb 16F4</td><td>N/A</td><td>N/A</td><td>raised against the purified HP-NAP of H. pylori strain NCTC 11637 as described in Refernce 23;&amp;nbsp; A gift from Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA&lt;br/&gt; http://www.sciencedirect.com/science/article/pii/S0264410X10017585</td></tr><tr><td>HiLoad 16/600 Superdex 200 pg</td><td>GE Healthcare Life Sciences</td><td>28989335</td><td>for gel filtration chromatography&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28989335</td></tr><tr><td>PlusOne silver staining kit, protein</td><td>GE Healthcare Life Sciences</td><td>17-1150-01</td><td>http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/17115001</td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare Life Sciences</td><td>17-1440-02</td><td>for density gradient centrifugation to purify human neutrophils&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-tw/products/AlternativeProductStructure&lt;br/&gt;_16963/17144002</td></tr><tr><td>Flat bottom 96-well white plate</td><td>Thermo Fisher Scientific Inc</td><td>236108</td><td>http://www.thermoscientific.com/en/product/nunc-f96-microwell-black-white-polystyrene-plate.html</td></tr><tr><td>Luminol</td><td>Sigma-Aldrich</td><td>A8511</td><td>protected from light&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/a8511?lang=en&amp;amp;region=TW</td></tr><tr><td>Expand &amp;nbsp;long template PCR system</td><td>Sigma-Aldrich</td><td>11681834001</td><td>source of High Fidelity PCR enzyme mix&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/roche/elongro?lang=en&amp;amp;region=TW</td></tr><tr><td>Dpn I</td><td>New England Biolabs</td><td>R0176S</td><td>https://www.neb.com/products/r0176-dpni</td></tr><tr><td>Xho I</td><td>New England Biolabs</td><td>R0146S</td><td>https://www.neb.com/products/r0146-xhoi</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; DH5&amp;alpha;</td><td>Thermo Fisher Scientific Inc</td><td>18265-017</td><td>https://www.thermofisher.com/order/catalog/product/18265017</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Product Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td>&lt;br/&gt; &amp;nbsp;</td></tr><tr><td>U-2800 double beam UV/VIS spectrophotometer</td><td>Hitachi&amp;nbsp;</td><td>N/A</td><td>out of market and upgraded to a new model&lt;br/&gt; http://hitachi-hta.com/products/life-sciences-chemical-analysis/uvvisible-spectrophotometers</td></tr><tr><td>EmulsiFlex-C3 high pressure homogenizer</td><td>Avestin Inc</td><td>C315320</td><td>http://www.avestin.com/English/c3page.html</td></tr><tr><td>Hitachi Koki himac CP80WX general ultracentrifuge</td><td>Hitachi Koki Co</td><td>90106401</td><td>for separation of the soluble and insoluble protein fractions from &lt;em&gt;E. coli&lt;/em&gt; lysates&lt;br/&gt; http://centrifuges.hitachi-koki.com/products/ultra/cp_wx/cp_wx.html</td></tr><tr><td>&amp;Auml;KTA FPLC</td><td>GE Healthcare Life Sciences</td><td>18-1900-26</td><td>for gel filtration chromatography&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/18190026</td></tr><tr><td>Aviv model 62ADS CD spectrophotometer</td><td>Aviv Biomedical</td><td>N/A</td><td>out of market and upgraded to a new model http://www.avivbiomedical.com/circular.php</td></tr><tr><td>LAS-3000 imaging system</td><td>Fujifilm</td><td>N/A</td><td>discontinued and replaced&lt;br/&gt; http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28955810</td></tr><tr><td>Wallac 1420 (Victor2) multilabel counter</td><td>Perkin-Elmer</td><td>1420-018</td><td>for chemiluminescence detection&lt;br/&gt; http://www.perkinelmer.com/catalog/product/id/1420-018</td></tr></tbody></table>

one-step negative chromatographic purification of helicobacter pylori neutrophil-activating protein overexpressed in escherichia coli in batch mode

幽门螺杆菌中性粒细胞激活蛋白（HP-NAP）是幽门螺杆菌 （ 幽门螺旋杆菌 ）的主要致病因子。它在H的关键作用幽门通过激活几个固有的白细胞包括嗜中性粒细胞，单核细胞和肥大细胞诱导的胃窦炎。 HP-NAP的免疫原性和免疫调节性质使其成为H的潜在的诊断和疫苗候选幽门和肿瘤治疗的新的候选药物。为了获得用于其临床应用纯化的HP-NAP实质性数量时，一个有效的方法，以纯化该蛋白质具有高产率和高纯度的需要建立。 在这个协议中，我们已使用二乙基氨基乙基（DEAE） -离子交换树脂中描述的方法用于在大肠杆菌 （ 大肠杆菌 ）中过表达重组的HP-NAP一步法阴性色谱纯化（ 例如 ，交联葡聚糖）我ñ批处理模式。重组的HP-NAP构成近70％在大肠杆菌中总蛋白的大肠杆菌和几乎完全在细胞裂解的可溶部分在pH9.0回收。下，在pH为8.0的最佳条件下，大多数的HP-NAP在未结合部分被回收，同时从大肠杆菌的内源性蛋白大肠杆菌是由树脂有效地除去。 使用负模式间歇色谱用DEAE离子交换树脂此纯化方法产生来自大肠杆菌官能的HP-NAP 大肠杆菌在高产量和纯度的天然形式。纯化的HP-NAP可进一步用于预防，治疗，及H的预后幽门机相关的疾病以及癌症的治疗。

重组的HP-NAP的负纯化的实验程序的示意图在大肠杆菌表达大肠杆菌通过在分批模式下使用DEAE离子交换树脂是在图1所示。该纯化技术是基于结合宿主细胞蛋白质和/或比的HP-NAP至树脂等杂质。在pH 8.0，除了在其天然形式的HP-NAP几乎没有其他的蛋白质与未结合的级分（ 图2A和B）回收。纯化的HP-NAP中未结合的级分通过依靠HP-NAP...

Watch this Scientific Journal Video about One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode at JoVE.co

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

用于重组幽门螺杆菌一步法负纯化的高产量方法，通过使用被描述在批处理模式二乙氨基树脂幽门在大肠杆菌中过表达嗜中性粒细胞激活蛋白（HP-NAP）。通过该方法纯化的HP-NAP是疫苗，药物或诊断为H的发展有利幽门螺杆菌机相关的疾病。

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

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Immunology and Infection

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

9.6K 次观看.  National Tsing Hua University. 用于重组幽门螺杆菌一步法负纯化的高产量方法，通过使用被描述在批处理模式二乙氨基树脂幽门在大肠杆菌中过表达嗜中性粒细胞激活蛋白（HP-NAP）。通过该方法纯化的HP-NAP是疫苗，药物或诊断为H的发展有利幽门螺杆菌机相关的疾病。 

视频: One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

科研

生物化学

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104纯化，蛋白质Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

生物学

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

免疫与感染

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

细菌中无机聚磷酸盐的检测

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

基于 qPCR 的快速、无创幽门 螺杆菌 感染和抗生素耐药性诊断

Quantitative Polymerase Chain Reaction (qPCR)-Based Rapid Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to its increasing antibiotic resistance. It is the causative agent of chronic active gastritis, peptic ulcer disease, and gastric cancer and has been classified as a Group I Carcinogen by the International Agency for Research on Cancer. Therefore, the rapid and accurate diagnosis of H. pylori and the determination of its antibiotic resistance are important for the efficient eradication of this bacterial pathogen. Currently, H. pylori diagnosis methods mainly include the urea breath test (UBT), the antigen test, the serum antibody test, gastroscopy, the rapid urease test (RUT), and bacterial culture. Among them, the first three detection methods are noninvasive, meaning they are easy tests to conduct. However, bacteria cannot be retrieved through these techniques; thus, drug resistance testing cannot be performed. The last three are invasive examinations, but they are costly, require high skills, and have the potential to cause damage to patients. Therefore, a noninvasive, rapid, and simultaneous method for H. pylori detection and drug resistance testing is very important for efficiently eradicating H. pylori in clinical practice. This protocol aims to present a specific procedure involving the string test in combination with quantitative polymerase chain reaction (qPCR) for the rapid detection of H. pylori infection and antibiotic resistance. Unlike bacterial cultures, this method allows for easy, rapid, noninvasive diagnosis of H. pylori infection status and drug resistance. Specifically, we used qPCR to detect rea for H. pylori infection and mutations in the 23S rRNA and gyrA genes, which encode resistance against clarithromycin and levofloxacin, respectively. Compared to routinely used culturing techniques, this protocol provides a noninvasive, low-cost, and time-saving technique to detect H. pylori infection and determine its antibiotic resistance using qPCR.

作者聚焦: 幽门螺杆菌感染的检测和治疗 

The protocol presents a noninvasive method for the rapid diagnosis of Helicobacter pylori stomach infections through the string test and determines its antibiotic resistance to clarithromycin and levofloxacin using quantitative polymerase chain reaction (qPCR).

基于定量聚合酶链反应（qPCR）的幽 门螺杆菌 感染和抗生素耐药快速诊断

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to ...

一步法负色谱纯化

摘要

探索更多视频

此视频中的章节

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

Hsp104纯化，蛋白质Disaggregase

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

细菌中无机聚磷酸盐的检测

高通量的亲和标签的重组蛋白的纯化

基于定量聚合酶链反应（qPCR）的幽门螺杆菌感染和抗生素耐药快速诊断

基于定量聚合酶链反应（qPCR）的幽门螺杆菌感染和抗生素耐药快速诊断

基于定量聚合酶链反应（qPCR）的幽门螺杆菌感染和抗生素耐药快速诊断

一步法负色谱纯化

摘要

探索更多视频

此视频中的章节

无核酸酶的表达和纯化原氧基质 3，4-二氧酶

使用多柱板适配器的经济性和多功能性高通量蛋白质净化系统

Hsp104纯化，蛋白质Disaggregase

由一个小型的双特异性亲和标签的推动正交蛋白质纯化

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

细菌中无机聚磷酸盐的检测

高通量的亲和标签的重组蛋白的纯化

基于定量聚合酶链反应（qPCR）的幽 门螺杆菌 感染和抗生素耐药快速诊断

基于定量聚合酶链反应（qPCR）的幽 门螺杆菌 感染和抗生素耐药快速诊断

基于定量聚合酶链反应（qPCR）的幽 门螺杆菌 感染和抗生素耐药快速诊断

基于定量聚合酶链反应（qPCR）的幽门螺杆菌感染和抗生素耐药快速诊断

基于定量聚合酶链反应（qPCR）的幽门螺杆菌感染和抗生素耐药快速诊断

基于定量聚合酶链反应（qPCR）的幽门螺杆菌感染和抗生素耐药快速诊断