这项研究得到了退伍军人事务部职业发展奖1IK2BX001701，国家推进转化科学中心的CTSA奖UL1TR000445，国家科学基金会奖1547757和1400969以及NIH授予GM05551的支持。其内容完全是作者的责任，不一定代表国家推进转化科学中心或国家卫生研究院的官方观点。

S100A12的表达<ol><li>使用标准热休克方案，用pGEMEX-S100a12质粒转化感受态BL21 DE3细胞。在微量离心管中在冰上向50μL细菌中加入1至5μL质粒。孵育20分钟</li><li>在42℃下将细胞热冲击30秒。 </li><li>将细胞在冰上孵育2分钟。 </li><li>向细胞中加入500μLSOC介质。在37℃下，在轨道振荡器上以250rpm振荡孵育1小时。 </li><li>在LB-琼脂培养基（补充100μg/ mL氨苄青霉素）上150μL的转化反应。在37°C孵育12-16 h。 </li><li>挑一个殖民地接种2 mL LB（补充100μg/ mL氨苄青霉素）。 </li><li>在37℃下在轨道振荡器（300rpm）上孵育4-6小时。 OD 600应读数在1-3个吸光度单位之间。 </li><li>加入500μL起始培养物至50 mL ZYM-5052 autoindu补充有100μg/ mL氨苄青霉素的补充培养基26 。为了获得最佳曝气和最大表达，请使用250 mL带挡板的锥形瓶。在37℃下摇动（300rpm）24小时。 </li><li>将细菌悬浮液转移到离心管中。在4℃下通过离心（4,000 xg，10分钟）将细胞造粒。 </li><li>将介质倾倒，记录样品，并将细胞粘贴存储在-80°C。样品多年稳定。 </li></ol> 2.使用低压色谱法纯化S100A12 <ol><li>将细胞重悬于30 mL 20 mM Tris，pH 8.0中。 </li><li>在冰上超声处理悬浮液以溶解细胞。使用〜20 W输出，5 s on和5 s off循环5 min。 </li><li>将溶液转移到高速离心管中。通过离心澄清细胞裂解物，在4℃下20,000×g 30分钟。 </li><li>倾析上清液并转移到干净的100mL聚丙烯烧杯中。通过将烧杯放在冰上来冷却溶液。添加一个搅拌缸并慢慢加入11.20g硫酸铵。让溶液在冰上再搅拌1小时。这将产生60％的硫酸铵溶液并沉淀大部分大肠杆菌内源性蛋白质。 S100A12将保持溶解。 </li><li>在4℃，20,000×g离心20分钟以沉淀沉淀的蛋白质。 </li><li>倾倒上清液并转移到透析管（MWCO 3,500 kDa）。在4℃下对1μL的20mM Tris，pH 8.0进行透析。更换透析缓冲液两次。允许4小时之间的变化。 </li><li> 阴离子交换层析 <ol><li>在低压系统上进行色谱。典型流速为1 mL / min。 </li><li>用10mL 20mM Tris，pH 8.0平衡5mL Sepharose柱。 </li><li>使用样品泵装载〜40 mL的S100A12溶液（收集流量）。 </li><li>用10mL 20mM Tris，pH8洗涤柱。 </li><li>用0-30％梯度（Bub B为20mM Tris，pH 8.0,1M NaCl），经19柱体积（CV，95mL）。收集5 mL级分。 </li><li>取每个级分的10μL等分试样，并用考马斯染色的MES SDS PAGE进行分析。使用恒定电压（20 V / cm）运行凝胶30分钟。 </li><li>含有S100A12的游泳池部分。 S100A12在变性凝胶上以约10kDa的蛋白质运行。 </li><li>使用超滤装置（MWCO 10kDa）浓缩至5mL。离心机在3,000 xg〜8分钟。取最高分数。 </li></ol></li><li> 大小排阻层析 <ol><li>用1 CV（120 mL）的20mM Tris pH8,100mM NaCl平衡S75柱。 </li><li>注入&lt;5 mL浓缩的S100A12级分（来自离子交换层析）。 </li><li>以120mL的流速以1mL / m的流速开发柱。收集5 mL级分。 </li><li>取每个级分的10μL等分试样，并用考马斯染色的SDS PAGE进行分析。使用蛋白质鉴定蛋白质印迹或质谱（计算的单体亚基的分子量为10,575.0Da，测量值为10575.4Da）。 </li></ol></li><li> 池分数 <ol><li>使用分光光度计测量蛋白A 280的吸光度。使用SEC缓冲区作为空白。计算出的S100A12同型二聚体的消光系数为5960M - 1cm -1 。 注意:每50 mL培养物的典型产量为35-45 mg的S100A12。 </li><li>将等分试样S100A12放入1.5 mL微量离心管（1 mg /管）中，在液氮中快速冷冻，储存于-80°C。 </li></ol></li></ol> 3.抗菌活性测定<ol><li>将条纹幽门螺杆菌菌株G27置于补充有5％绵羊血液（血琼脂平板）的胰蛋白酶大豆琼脂平板上。在37℃下在补充有5％二氧化碳的室内空气中生长2-3天。 </li><li>将幽门螺杆菌接种到补充有1x胆固醇的布鲁杆菌肉汤中。文化过夜间在37℃下，在补充有5％二氧化碳的室内空气中摇动（250rpm）。 </li><li>将幽门螺杆菌 1:10稀释成50％布鲁氏菌肉汤，50％钙卫蛋白缓冲液加1x胆固醇，培养液中单独培养，或补充100μM氯化锌加0,100或1000μg/ mL纯化的S100A12。培养过夜振荡（250rpm），37℃，室温下补充5％二氧化碳。 </li><li>第二天，连续稀释并平板至血琼脂平板上。允许细菌菌落在37℃下在补充有5％二氧化碳的室内空气中生长2-3天。在存在或不存在S100A12和/或外源性锌的情况下，列举菌落形成单位以计算细菌生长。 </li></ol>

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<li>Goyette, J., Geczy, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammation-associated+S100+proteins%3A+new+mechanisms+that+regulate+function%5BTitle%5D)+AND+%22Amino+Acids%22%5BJournal%5D)">Inflammation-associated S100 proteins: new mechanisms that regulate function.</a> Amino Acids. 41 (4), 821-842 (2011).</li>
<li>Kehl-Fie, T. E., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nutritional+immunity+beyond+iron%3A+a+role+for+manganese+and+zinc%5BTitle%5D)+AND+%22Curr+Opin+Chem+Biol%22%5BJournal%5D)">Nutritional immunity beyond iron: a role for manganese and zinc.</a> Curr Opin Chem Biol. 14 (2), 218-224 (2010).</li>
<li>Zackular, J. P., Chazin, W. J., Skaar, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nutritional+Immunity%3A+S100+Proteins+at+the+Host-Pathogen+Interface%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Nutritional Immunity: S100 Proteins at the Host-Pathogen Interface.</a> J Biol Chem. 290 (31), 18991-18998 (2015).</li>
<li>Realegeno, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((S100A12+Is+Part+of+the+Antimicrobial+Network+against+Mycobacterium+leprae+in+Human+Macrophages%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">S100A12 Is Part of the Antimicrobial Network against Mycobacterium leprae in Human Macrophages.</a> PLoS Pathog. 12 (6), e1005705(2016).</li>
<li>Perera, C., McNeil, H. P., Geczy, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((S100+Calgranulins+in+inflammatory+arthritis%5BTitle%5D)+AND+%22Immunol+Cell+Biol%22%5BJournal%5D)">S100 Calgranulins in inflammatory arthritis.</a> Immunol Cell Biol. 88 (1), 41-49 (2010).</li>
<li>Moroz, O. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+the+human+S100A12-copper+complex%3A+implications+for+host-parasite+defence%5BTitle%5D)+AND+%22Acta+Crystallogr+D+Biol+Crystallogr%22%5BJournal%5D)">Structure of the human S100A12-copper complex: implications for host-parasite defence.</a> Acta Crystallogr D Biol Crystallogr. 59 (Pt 5), 859-867 (2003).</li>
<li>Moroz, O. V., Blagova, E. V., Wilkinson, A. J., Wilson, K. S., Bronstein, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+crystal+structures+of+human+S100A12+in+apo+form+and+in+complex+with+zinc%3A+new+insights+into+S100A12+oligomerisation%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">The crystal structures of human S100A12 in apo form and in complex with zinc: new insights into S100A12 oligomerisation.</a> J Mol Biol. 391 (3), 536-551 (2009).</li>
<li>Moroz, O. V., Dodson, G. G., Wilson, K. S., Lukanidin, E., Bronstein, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+structural+states+of+S100A12%3A+A+key+to+its+functional+diversity%5BTitle%5D)+AND+%22Microsc+Res+Tech%22%5BJournal%5D)">Multiple structural states of S100A12: A key to its functional diversity.</a> Microsc Res Tech. 60 (6), 581-592 (2003).</li>
<li>Cunden, L. S., Gaillard, A., Nolan, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+Ions+Tune+the+Zinc-Sequestering+Properties+and+Antimicrobial+Activity+of+Human+S100A12%5BTitle%5D)+AND+%22Chem+Sci%22%5BJournal%5D)">Calcium Ions Tune the Zinc-Sequestering Properties and Antimicrobial Activity of Human S100A12.</a> Chem Sci. 7 (2), 1338-1348 (2016).</li>
<li>Haley, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Human+Antimicrobial+Protein+Calgranulin+C+Participates+in+Control+of+Helicobacter+pylori+Growth+and+Regulation+of+Virulence%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence.</a> Infect Immun. 83 (7), 2944-2956 (2015).</li>
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<li>Leach, S. T., Mitchell, H. M., Geczy, C. L., Sherman, P. M., Day, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((S100+calgranulin+proteins+S100A8%2C+S100A9+and+S100A12+are+expressed+in+the+inflamed+gastric+mucosa+of+Helicobacter+pylori-infected+children%5BTitle%5D)+AND+%22Can+J+Gastroenterol%22%5BJournal%5D)">S100 calgranulin proteins S100A8, S100A9 and S100A12 are expressed in the inflamed gastric mucosa of Helicobacter pylori-infected children.</a> Can J Gastroenterol. 22 (5), 461-464 (2008).</li>
<li>Wilkie-Grantham, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloperoxidase-dependent+lipid+peroxidation+promotes+the+oxidative+modification+of+cytosolic+proteins+in+phagocytic+neutrophils%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Myeloperoxidase-dependent lipid peroxidation promotes the oxidative modification of cytosolic proteins in phagocytic neutrophils.</a> J Biol Chem. 290 (15), 9896-9905 (2015).</li>
</ol>

作者宣称他们没有竞争的经济利益。

提出了一种用于人S100A12的表达和纯化的有效方案。 大肠杆菌表达系统是用于生产重组蛋白的最常用工具，特别是生物化学和生物物理学研究需要mg量时。这里描述的程序的重要增强是使用自动诱导培养基26 ，其与标准的Luria-broth培养基18的表达相比将纯化蛋白的产量提高了近三十倍。另外，自动感应介质大大简化了蛋白质表达工作流程。使用常规生长培?...

S100蛋白是一类具有多种功能的钙结合蛋白的EF手系家族1 。它们以组织和细胞特异性方式表达，并调节广泛的细胞功能2,3 。钙结合蛋白独特，S100蛋白表现出细胞内和细胞外功能4,5 。在细胞内，Ca 2+结合诱导构象变化，其暴露出特异性靶向蛋白质结合配偶体的疏水性表面。这种细胞内机制调节重要的过程，如细胞增殖，分化和能量代谢。在细胞外环境中，S100蛋白表现出两个功能7 。一方面，它们作为损伤相关分子模式（DAMP）蛋白质，并引发促炎症通过与模式识别受体8,9的相互作用产生免疫反应。此外，S100蛋白质类螯合物的几个成员过渡金属，这是一种功能，用于在称为营养免疫的过程中将微生物病原体饥饿10,11 。 S100A12（也称为钙粒蛋白C和EN-RAGE）在巨噬细胞和嗜中性粒细胞中高度表达，并且已被鉴定为炎性疾病的潜在生物标志物12,13 。除了在其EF手部位点处结合钙之外，S100A12还具有位于二聚体界面14,15相对端的两个高亲和力的过渡金属结合位点。每个结合位点由三个组氨酸残基和一个天冬氨酸残基组成，可螯合锌或铜sup class ="xref"&gt; 16,17。最近，我们报道S100A12依赖性锌饥饿在调节幽门螺杆菌生长和促炎毒力因子的活性中是重要的18 。 幽门螺杆菌感染世界人口约一半的胃;使其成为最成功的细菌病原体之一19 。 幽门螺杆菌感染可导致胃炎，胃肠炎，十二指肠溃疡，粘膜相关淋巴组织（MALT）淋巴瘤，侵袭性胃腺癌（胃癌）等胃肠疾病。胃癌是世界上非贲门癌相关死亡的主要原因，胃癌的单一最大相关危险因素是幽门螺杆菌感染 。 幽门螺杆菌仍然存在于胃的利基，尽管如此对病原体的免疫应答，强调需要更好地了解控制这种细菌感染的免疫机制20,21,22,23。 幽门螺杆菌相关炎症的特征在于多形核细胞或嗜中性粒细胞的深度浸润，其在感染部位18,24,25上沉积抗微生物蛋白质（包括S100A12）的所有组成成分。为了了解宿主和病原体之间的复杂对话，我们力求改进纯化S100A12的技术，并用它来研究其对该医学相关病原体的抗菌作用。下面的方案概述了其生物活性状态下S100A12纯化的改进技术;能够与高亲和力结合营养金属并将它们从侵入的微生物中螯合。此外，以下方法突出了该蛋白质作为关键试剂的用途，以研究天然抗微生物分子限制细菌病原体生长的机制。 S100A家族蛋白作为参与免疫信号传导和宿主防御的重要的先天免疫系统分子群已经获得了欣赏27 。其中最为深入的研究是钙卫蛋白（MRP-8/14，钙粒蛋白A / B，S100A8 / A9） 28,29,30 。钙卫蛋白是中性粒细胞相关蛋白，其形成S100A8和S100A9亚基的异二聚体，其在二聚体界面31处结合过渡金属。钙卫蛋白已显示具有两个金属结合位点:位点1可以结合Zn 2+ ，Mn 2+或Fe 2+ ，而Site 2可以<html>结合Zn 2+ 31,32 。许多报告已经证明，钙卫蛋白具有抗多种病原体的抗菌活性 ， 包括金黄色葡萄球菌 ， 白色念珠菌 ， 鲍曼不 动杆菌 ， 肺炎克雷伯杆菌 ， 大肠杆菌和幽门螺杆菌 ，并且抑制作用是由于钙卫蛋白25,28的金属螯合活性， 29 。 以前的工作证明钙卫蛋白在幽门螺杆菌上发挥了许多活性，包括改变外膜中的脂质A结构，抑制cag- IV型分泌系统（其是幽门螺杆菌中的主要促炎毒力因子），诱导生物膜形成和抑制幽门螺杆菌生长和活力以剂量依赖的方式class ="xref"&gt; 25，33。此外，遗传和生物化学测定显示钙卫蛋白对幽门螺杆菌的抗菌活性主要来源于其与营养锌25的结合能力。 幽门螺杆菌需要锌，如先前的研究所确定的，其利用化学成分确定的培养基来确定该病原体生长和增殖的微量营养素需求34 。此外，钙卫蛋白在幽门螺杆菌感染的组织中高度丰富，并与嗜中性粒细胞浸润相关，表明宿主可能在感染和随后的炎症中使用钙卫蛋白作为抗微生物策略。 来自无偏蛋白质组学筛选技术的最新证据表明，在活性氧丰富的条件下，calprotec锡经历改变六组氨酸结合位点的翻译后修饰，从而抑制蛋白36的金属结合活性。因此，我们假设在这些分子的广泛谱系中的其他S100A家族蛋白可能潜在地作为辅助金属螯合剂。我们选择S100A12进行进一步研究，因为在上述翻译后修饰屏幕中没有鉴定，它具有结合锌的能力，并且在来自幽门螺旋杆菌感染个体的人体组织中高度丰富。 我们的工作表明，S100A12可以在幽门螺杆菌 G27菌株中以剂量依赖的方式抑制幽门螺杆菌生长和活力，并且通过添加过量的营养锌可以逆转该蛋白的抗微生物活性。这项工作补充了我们以前的工作，表明S100A12对PMSS1和7.1产生抗菌活性3株幽门螺杆菌 ，证明其对许多临床分离株和实验室适应的幽门螺杆菌菌株18的广谱抗菌活性。一起，这些结果证实了S100A12作为通过营养免疫来控制细菌生长和增殖的机制的重要性。未来对这种重要的宿主 - 细菌相互作用的研究可能包括利用S100A12的活性来减少宿主组织内的细菌负担，或者确定这种蛋白质在幽门螺旋杆菌感染背景下对免疫信号传导的贡献。

<table><tbody><tr><td>&lt;strong&gt;S100A12 Expression&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Expression cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BL 21 (DE3) competent cells</td><td>New England Biolabs</td><td>C25271</td><td></td></tr><tr><td>Autoinduction medium components</td><td></td><td></td><td></td></tr><tr><td>Yeast Extract</td><td>Research Products International</td><td>Y20020-250.0</td><td></td></tr><tr><td>NaCl</td><td>Research Products International</td><td>S23020-500.0</td><td></td></tr><tr><td>Tryptone</td><td>Research Products International</td><td>T60060-250.0</td><td></td></tr><tr><td>FeCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>F2877</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>M65240-100.0</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>S23100-500.0</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>P41200-500.0</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Research Products International</td><td>A20424-500.0</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Research Products International</td><td>S25150-500.0</td><td></td></tr><tr><td>Glycerol</td><td>Research Products International</td><td>G22020-1.0</td><td></td></tr><tr><td>D-glucose</td><td>Research Products International</td><td>G32040-500.0</td><td></td></tr><tr><td>lactose</td><td>Sigma-Aldrich</td><td>L2643</td><td></td></tr><tr><td>&lt;strong&gt;Selection agent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ampicillin</td><td>Research Products International</td><td>A40040-5.0</td><td></td></tr><tr><td>&lt;strong&gt;S100A12 Purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AKTA Start Chromatography System</td><td>GE</td><td>29-220-94</td><td></td></tr><tr><td>HiTrap Q Sepharaose Fast Flow</td><td>GE</td><td>17-5156-01</td><td></td></tr><tr><td>HiPrep 16/60 S-200 HR</td><td>GE</td><td>17-1166-01</td><td></td></tr><tr><td>Nanodrop lite spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-LITE</td><td></td></tr><tr><td>Tris Base</td><td>Research Products International</td><td>T60040-1000.0</td><td></td></tr><tr><td>&lt;em&gt;H. pylori &lt;/em&gt;Culture</td><td></td><td></td><td></td></tr><tr><td>Blood agar plates</td><td>Lab Supply Company</td><td>BBL221261</td><td></td></tr><tr><td>Brucella broth</td><td>Sigma-Aldrich</td><td>B3051</td><td></td></tr><tr><td>Cholesterol (250x)</td><td>Thermo Fisher Scientific</td><td>12531018</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>793566</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Zinc Chloride</td><td>Sigma-Aldrich</td><td>229997</td><td></td></tr></tbody></table>

expression, purification, and antimicrobial activity of s100a12

钙粒蛋白蛋白是先天免疫的重要介质，是钙结合蛋白的EF手家族的S100类的成员。一些S100蛋白质具有以高亲和力结合过渡金属的能力，并且在称为"营养免疫"的过程中有效地将其远离入侵的微生物病原体。 S100A12（EN-RAGE）结合锌和铜，并且在先天免疫细胞如巨噬细胞和嗜中性粒细胞中是高度丰富的。我们报告了一种用于表达，富集和纯化其活性金属结合构型的S100A12的精制方法。这种蛋白质在细菌生长和活力分析中的应用揭示了S100A12对细菌病原体幽门螺杆菌具有抗菌活性。抗菌活性取决于S100A12的锌结合活性，S100A12螯合营养锌，从而使需要锌生长的幽门螺杆菌饥饿roliferation。

S100A12表达纯化三步纯化从50mL细菌培养物中产生约40mg的重组S100A12。第一步是内源性大肠杆菌蛋白质的硫酸铵沉淀。该步骤之后是阴离子交换层析（ 图 1A ）。通过用考马斯亮蓝染色的SDS-PAGE追踪蛋白质（ 图1B ）。纯化方法的最后一步是将含有S100A12的合并级分用于大小排阻色谱法，用?...

Watch this Scientific Journal Video about Expression, Purification, and Antimicrobial Activity of S100A12 at JoVE.com

Expression, Purification, and Antimicrobial Activity of S100A12

在这里，我们提出了一种表达和纯化S100A12（钙粒蛋白C）的方法。我们描述了一种测定其对人类幽门螺杆菌的抗微生物活性的方案 。

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

expression-purification-and-antimicrobial-activity-of-s100a12

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8.3K 次观看.  Fisk University. 在这里，我们提出了一种表达和纯化S100A12（钙粒蛋白C）的方法。我们描述了一种测定其对人类幽门螺杆菌的抗微生物活性的方案 。 

视频: Expression, Purification, and Antimicrobial Activity of S100A12

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

亲和色谱法表达和纯化人基质金属蛋白酶-3

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

科研

生物化学

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

研究鞘氨醇1-磷酸受体结构和信号通路的管道

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

免疫与感染

通过亲和色谱从 Sipunculus nudus 中纯化纤溶酶

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and&#160;19,200 U/mL,&#160;respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

作者聚焦：从 Sipunculus nudus 中亲和纯化纤溶酶  

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

从海蛾中亲和纯化纤维蛋白溶解酶

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

生物工程

Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for immune cell stimulations. This protocol is based on a two-step chromatography strategy, which comprises protein pre-purification on an anion-exchange chromatography column and a subsequent polishing step on a hydrophobic-interaction column. This strategy produces S100A12 protein of high purity and yield at manageable costs. For functional assays on immune cells eventual remnant endotoxin contamination requires careful monitoring and further cleaning steps to obtain endotoxin-free protein. The majority of endotoxin contaminations can be excluded by anion-exchange chromatography. To deplete residual contaminations, this protocol describes a removal step with centrifugal filters. Depending on the available ion-strength S100A12 can arrange into different homomultimers. To investigate the relationship between structure and function, this protocol further describes ion-treatment of S100A12 protein followed by chemical crosslinking to stabilize S100A12 oligomers and their subsequent separation by size-exclusion chromatography. Finally, we describe a cell-based assay that confirms the biological activity of the purified protein and confirms LPS-free preparation.

This protocol describes a purification method for recombinant tag-free calcium binding protein S100A12 and its ion-induced oligomers for human monocyte stimulation assays.

人类S100A12及其用于免疫细胞刺激的Ion诱导寡聚物的纯化

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

生物学

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

S100A12的表达，纯化和抗菌活性

摘要

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人类S100A12及其用于免疫细胞刺激的Ion诱导寡聚物的纯化

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化