我们承认对他的支持和对他们的建议和意见，诺姆Grimberg Geula达维多夫和陈格特曼博士阿米尔Aharoni。

1。 妈妈在大肠杆菌中的基因的克隆和表达大肠杆菌 突变基因mamAΔ41Magnetospirillum magneticum AMB - 1的基因组DNA扩增的聚合酶链反应（PCR）引物：5&#39; - GCATTACGCATATGGACGACATCCGCCAGGTG 3&#39;和5&#39; - GCGCGGCAGCCATA - TGGCATACG - 3“ 。在扩增的DNA片段，NcoI网站介绍，在起始密码子ATG和终止密码子与ScoI网站所取代。片段，NcoI和SACI消化和克隆到pET52b（+），在各自的网站，从而引发pET52bMamAΔ41- AMB1。在这种构造，mamAΔ41基因融合在帧与10他在C -末端的标记。质粒电穿孔转化大肠杆菌菌株BL21。大肠杆菌菌株BL21窝藏pET52bMamAΔ41自动感应（15）生长在培养基中含有氨苄青霉素（50毫克/毫升）310 ° K时为3小时。然后转向培养温度从310 °到300 ° K的和额外的48小时保持在300 ° K。收获细胞，在5465克离心10分钟在277 ° K。 8升文化生产的60克湿细胞沉淀。 2。生物信息学的计算分子量（MW），根据氨基酸序列预测蛋白质在280nm处1mg/1ml吸收计算，使用ProtParam服务器（http://www.expasy.ch/tools/protparam.html）。对于10His标签MamAΔ41兆瓦22529大（240个氨基酸）和MamAΔ41（His - tag的凝血酶搬迁后留下的9个氨基酸）兆瓦20596.5Da（187个氨基酸）。 1mg/1ml预测10His标签MamAΔ41的蛋白质在280nm处的吸收是0.595和MamAΔ41是0.579。此外，氨基酸序列不包含任何半胱氨酸残基。因此不需要在净化过程中，还原剂。 3。纯化的MamAΔ41 <ol><li>储备液的制备，掂量出，并准备在下面的浓度（每200毫升）：咪唑（兆瓦228.2克/摩尔）4M的解决方案，氯化钠（MW 58.44克/摩尔）5M解决方案的Tris - HCl（分子量121.3克/摩尔）1M溶液，调节pH = 8。加入双蒸水（DDW）和混合使用的解决方案是一个旋涡，直到清晰。过滤0.22μm的过滤器，并保持在室温解决方案。 </li><li>准备和筛选股票的解决方案（表1）新鲜缓冲区。 </li><li>稀释和缓冲暂停一个1:2的比例，细胞克缓冲体积（ml）的细胞。 </li><li>每个细菌的DNA酶I（1毫克/毫升）10gr新增加入10μl为了打破样品中的DNA片段。添加为每个20gr细菌无EDTA的蛋白酶抑制剂的鸡尾酒1毫升。孵育20分钟冰。 </li><li>打乱了两个周期的法国记者在25,000 psi的细胞。与超声不同的是，法国媒体的目的是通过薄的喷嘴在高压下，迫使细胞在高音量（&gt; 10毫升）细胞挤压。活塞应在冰上冷却10分钟前，其使用和装配，由于温度建设。 </li><li>转移到25毫升超速离心机管裂解细胞并平衡他们正是用缓冲液A（50毫克），直到样品达到管的脖子线。 </li><li>由1小时270,000克的超离心分离细胞碎片在277 ° K。 </li><li>准备一个自制的重力镍NTA柱（2.5厘米直径）4毫升镍NTA树脂，保留20％的乙醇，添加到列。完全让流体下降，然后用40毫升DDW，完全让流体下降。预平衡的树脂，用40毫升缓冲液A列</li><li>应用重力镍NTA柱的超离心管中的可溶部分。让液体滴，收集流量（未结合的蛋白质），并保持在277K。 </li><li>通过使用一个小技巧废料颗粒颗粒样品，收集从超离心管。在50μL的5％β-巯基乙醇在2X SDS - PAGE样品缓冲液混合小费。 </li><li> 100毫升缓冲B.清洗列，让液体滴，收集流通过，并保持在277K。 </li><li>准备15标记Eppendorf管（1.5毫升）。 </li><li>洗脱 - 关闭阀门，然后列列装载到缓冲液C1毫升; 3分钟流过阀门的开度和收集孵化。重复此步骤三次。 </li><li>洗脱其余悬浮于1 ml缓冲液C为增量和关闭阀门。 </li><li>测量的分数为280nm OD和评估的蛋白峰的位置;如果外径&gt; 0.3，需要额外的样品。 </li><li> SDS - PAGE分析收集馏分。应该进行这样的分析还与以下的样品，未结合的蛋白质流过，洗流和ULTRA离心沉淀样品。 10μL5％的2X SDS - PAGE样品缓冲液中的β-巯基乙醇混合从每个样品中加入10μl。负荷15％的SDS - PAGE的样品和运行在180伏特，45分钟。 </li><li>凝胶染色InstantBlue解决方案在15毫升，摇匀1小时。确保塑料盒覆盖避光。 </li><li>评估凝胶;如果蛋白表达的是高的和具体的指示带，合并选定的洗脱组分。 </li><li>为了消除10 - His - tag的，加入牛凝血酶（10单位/μL），加入10μl凝血酶每1毫克的蛋白质，根据制造商的建议合并范例。 </li><li>透析用于咪唑去除多余的，在离子交换绑定需要的水平，以减少NaCl浓度。透析，切成所需长度7 kD的分子量（MWCO）透析管切断。与DDW清洗，并在一端（或剪辑盖章）打了一个结。转移到合并后的蛋白质溶液袋，而在顶部留下一定的空间和遏止。 </li><li>淹没在4升的冷冻透析缓冲的透析管（缓冲液D）。搅拌的解决方案，在277 ° K.缓慢的夜晚</li><li>透析管出烧杯，使一小截的蛋白质转移至50ml管。 </li><li>离子交换色谱：组装快速高效液相色谱仪（FPLC）预先包装MonoQ 4.6/100 PE列。 DDW过滤后的水用5倍柱体积（CV）的清洗之列。平衡5 CV的缓冲区D.列</li><li>注射液循环与消化疾病周（两个循环卷）洗净，并用缓冲液D.重复负载在注射循环中的蛋白质。 </li><li>注入的蛋白质样品（流速为1毫升/分钟），并开始收集馏分无限蛋白进行SDS - PAGE检测。 </li><li>如果最初的蛋白量（步长3.23）大于注射循环，重复步骤3.25-3.26，不洗循环，直到整个样本列装载。 </li><li>为了消除无限的蛋白质，洗净后用缓冲液C CV 3列。 </li><li> 60分钟线性梯度之间的缓冲区D，E（100％缓冲液E是渐变结束时），流速为1毫升/分钟（40 2000MM氯化钠洗脱的蛋白质，虽然可以在成本较高的流速更大的卷，但不太集中，蛋白质峰）。收集蛋白峰在280nm处的色谱图中出现的。 </li><li>清洁的预包装列，根据生产的建议。 </li><li> SDS - PAGE分析收集馏分。这种分析应执行下列范例，无限的蛋白质流和蛋白峰流过。运行的SDS - PAGE和染色提步骤3.17-3.18。 </li><li>评估预测兆瓦凝胶对蛋白条带的存在，根据预染蛋白质标记。合并选定的洗脱组分包含有关的蛋白质。 </li><li>透析合并后的样品，以较低的体积排阻色谱所需的NaCl浓度。执行对缓冲区F.步骤3.21-3.23提到透析</li><li>浓缩的蛋白质样品使用一个Vivaspin 15万截留分子量为8毫克/毫升表中的离心机，4000转。验证愿望浓度石英比色皿在280nm处测量外径。如果过于集中的蛋白质与缓冲F稀释和测量一次。 </li><li>装配尺寸排阻预填充柱，HiLoad 26/60 Superdex 200，上FPLC;用3 DDW过滤水的简历列。平衡列缓冲区楼与3简历</li><li>其次是与相同体积的缓冲楼（两个循环卷）用5ml DDW清洗注射液循环的蛋白质样品注入回路中负载不超过4毫升。 </li><li>注入蛋白质样品（流速为3.5毫升/分钟），并开始收集蛋白峰分数，因为它们出现在280nm处的色谱。让缓冲区的流量，直至达到柱体积。 </li><li>如果最初的蛋白量（步长3.23）大于注射循环，重复步骤3.25-3.26，不洗循环，直到整个样本列装载。 </li><li>清洁的预包装列，根据生产的建议。 </li><li> SDS - PAGE分析收集馏分。这种分析应与蛋白质峰的样品。运行的SDS - PAGE和染色提步骤3.17-3.18。 </li><li>评估凝胶：如果蛋白峰是具体的，在正确预测兆瓦的根据预染蛋白质标记和只有一个乐队可见合并选定的相关蛋白的洗脱组分。 </li><li>浓缩蛋白样品使用一个Vivaspin 15万截留分子量大于20毫克/毫升表离心机，4000转。验证欲望的浓度，在280nm处测量外径。纯化MamAΔ41，然后集中到26.5毫克/毫升的结晶。 </li> &lt;李&gt;确认样品纯度和蛋白质鉴定，使用基质辅助激光解吸/电离（MALDI - TOF）。 </li><li>如果根据质谱仪和SDS - PAGE纯化的蛋白质，划分前显着的Eppendorf管集中MamAΔ41; 25 -50μL每个蛋白质。 </li><li>闪光冷冻在液氮和存储在193 ° K。 </li></ol> 4。代表性的成果当这个协议是正确完成的，应该得到高度纯化，大小一致和集中的蛋白质样品。这些蛋白质样品，然后准备进行结晶试验以及生化研究，如酶动力学，亲和力和更多。这里描述的是在净化协议的主要步骤代表性的结果。 NI - NTA亲和层析柱洗脱概况的SDS - PAGE分析，揭示过度表达蛋白可溶部分高度在适当兆瓦〜22kDa（图1）。这个SDS - PAGE分析也揭示最低，如果任何在流通过无界蛋白的蛋白质，洗流和超离心沉淀样品。如果适当的蛋白质的大型乐队出现在这些样品中，应该考虑错误的缓冲液配制，细胞破碎或文化的自动感应的条件和发展中的问题的问题。 离子交换层析是预制的，为了我们的理想蛋白质之间的单独其他大肠杆菌的蛋白质，势必镍树脂（由于静电相互作用或组氨酸/负富含氨基酸的蛋白质循环）和他的标签完整无缺的理想蛋白质。此列根据自己的亲和力增加NaCl浓度下正电的树脂分离蛋白。高度带负电荷的蛋白质会在较高浓度的NaCl洗脱放在附近，以温和的带负电荷的。本专栏的优点是高流速和有约束力的能力。离子交换色谱图（图2）揭示了3蛋白在NaCl浓度增加的人口之间的分离效果好。 SDS - PAGE分析，以确定每个人口兆瓦，隔离的理想之一，评估是否需要进一步的纯化步骤。第一人口MamAΔ41（〜20 kDa的），而第二人口MamAΔ41+他的标记（〜22kDa），这是没有牛凝血酶裂解和三分之一的人口是由于低浓度的SDS - PAGE检测不到。如果蛋白质峰没有分开，显然应该考虑错误的缓冲液配制或改变的NaCl梯度的斜率。 体积排阻色谱预制以之间的理想其他的大肠杆菌细胞，势必镍- NTA树脂，并没有在离子交换色谱分离蛋白的蛋白质分离。此柱分离蛋白质根据它们的大小。大的蛋白质会在较小的洗脱反对将在更大的卷洗脱小卷洗脱。柱色谱图（图3）揭示了一个理想的蛋白质作为一个主要的人口的分离效果好。一个兆瓦〜20 kDa的人口出现在适当的单体规模洗脱。 〜80 kDa的乐队（典型大肠杆菌的蛋白质结合Ni - NTA树脂的）的存在是在SDS - PAGE检测列装载之前和在此期间运行消失。由于本〜80 kDa的蛋白浓度低开始，它的人口不会出现在色谱由于其稀释和SDS - PAGE分析，以确定净化效率。我们建议主峰一个装载稀释和浓缩样品的SDS - PAGE，所以肯定可以决定在适当的兆瓦的纯蛋白质的存在。到了这个阶段，蛋白质纯化和其同质化应用MALDI - TOF评估。这分析显示，在20347达和其他10176大（图4）MW MW蛋白质。 〜10 kDa的蛋白〜20 kDa的蛋白一倍收取。 MamAΔ41预测兆瓦他的标签去除后留下的9个氨基酸为20596.5大。通过比较所获得的MALDI - TOF兆瓦，我们发现，他们是248大除。这种差异性可能导致的MALDI - TOF的测量误差和/或由于常见的退化的第一蛋氨酸和第二甘氨酸。总括而言，蛋白质是高度纯化的，可用于进一步的实验。 <img src="/files/ftp_upload/1844/1844table.jpg" alt="表1" /> 表1。缓冲液配方。 <img src="/files/ftp_upload/1844/1844fig1.jpg" alt="图1" /> 图1。代表SDS - PAGE分析NI - NTA柱纯化由右至左;的P -超速离心沉淀（3.11协议的步骤），W -洗（3.12协议的步骤），U -无限的蛋白质（3.10协议的步骤），E -五车道洗脱桑普LES（3.14协议的步骤），M - 蛋白标记（数字显示兆瓦）。箭头指示MamAΔ41。 <img src="/files/ftp_upload/1844/1844fig2.jpg" alt="图2" /> 图2。离子交换层析步骤 （一）离子交换（MonoQ柱）色谱分析 ;蓝色-吸收为280nm，蛋白浓度，红，绿NaCl浓度-收集分数的指示。 （b）代表SDS - PAGE分析的离子交换纯化步骤。从左至右依次为，M - 蛋白标记（数字表明MW），A6 - 第一峰的一小部分，A8的第二个高峰期的一小部分。 <img src="/files/ftp_upload/1844/1844fig3.jpg" alt="图3" /> 图3。体积排阻色谱分析 （一）排阻（Superdex 200柱）色谱图，蓝色-吸收为280nm，蛋白浓度，红色指示收集馏分。 （b）代表SDS - PAGE分析的体积排阻纯化步骤。从左至右依次为M蛋白标记（数字显示兆瓦），PreI预进样，A4 - 6组合的一小部分，从收集到的第一个高峰，A4 - 6D摊薄分数收集从高峰。 <img src="/files/ftp_upload/1844/1844fig4.jpg" alt="图4" /> 图4。基质辅助激光解吸/电离（MALDI - TOF）大规模纯化MamAΔ41频谱。矩阵是芥子酸（SA）。 MamAΔ4120347大，也显示一倍收取物种MamAΔ4110176大。

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein-import+machinery:+correlating+structure+with+function.">Mitochondrial protein-import machinery: correlating structure with function.</a> Trends Cell Biol. 17, 456-464 (2007).</li><li>Mirus, O., Bionda, T., von Haeseler, A., Schleiff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionarily+evolved+discriminators+in+the+3-TPR+domain+of+the+Toc64+family+involved+in+protein+translocation+at+the+outer+membrane+of+chloroplasts+and+mitochondria.">Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria.</a> J Mol Model. 15, 971-982 (2009).</li><li>Gatsos, X., Perry, A. J., Anwari, K., Dolezal, P., Wolynec, P. P., Likic, V. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetratricopeptide+repeats+in+the+type+III+secretion+chaperone,+LcrH:+their+role+in+substrate+binding+and+secretion.">Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.</a> Mol Microbiol. 59, 31-44 (2006).</li><li>Grunberg, K., Muller, E. C., Otto, A., Reszka, R., Linder, D., Kube, M., Reinhardt, R., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+proteomic+analysis+of+the+magnetosome+membrane+in+Magnetospirillum+gryphiswaldense.">Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense.</a> Appl Environ Microbiol. 70, 1040-1050 (2004).</li><li>Komeili, A., Vali, H., Beveridge, T. J., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosome+vesicles+are+present+before+magnetite+formation,+and+MamA+is+required+for+their+activation.">Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation.</a> Proc Natl Acad Sci USA. 101, 3839-3843 (2004).</li><li>Okuda, Y., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+a+magnetosome-associated+protein,+TPR-containing+MAM22,+in+Escherichia+coli.">Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli.</a> FEBS Lett. 491, 169-173 (2001).</li><li>Taoka, A., Asada, R., Sasaki, H., Anzawa, K., Wu, L. F., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localizations+of+Mam22+and+Mam12+in+the+magnetosomes+of+Magnetospirillum+magnetotacticum.">Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum.</a> J Bacteriol. 188, 3805-3812 (2006).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41, 207-234 (2005).</li></ol>

蛋白质纯化的生化或在任何蛋白质结构研究的主要步骤。由于每个蛋白质独特的是与自己的行为，需要定义其属性，并相应修改其净化。分析蛋白质的目标应该是，作为对使用净化生物信息学工具的第一步。它们被用来计算目标ISO电点，评估其需要减少/氧化环境，它需要特殊的离子/配体。我们的协议中有几个关键的修改反映了目标的独特性。这些修改包括缓冲区，工作温度和列调整。 <p class=...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French Press</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-078A</td>
<td>&nbsp;</td>
<tr>
<td>Pressure cell</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-032</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge</td>
<td>Equipment</td>
<td>Sorvall</td>
<td>Discovery 90SE</td>
<td>&nbsp;</td>
<tr>
<td>Rottor</td>
<td>Equipment</td>
<td>Beckman</td>
<td>Ti60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml</td>
<td>Equipment</td>
<td>Beckman</td>
<td>BC-355618</td>
<td>&nbsp;</td>
<tr>
<td>2.5cm diameter, Glass Econo-Column Chromatography Columns</td>
<td>Equipment</td>
<td>BioRad</td>
<td>737-2521</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA His Bind resin</td>
<td>Equipment</td>
<td>Novagen</td>
<td>M0063428</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer</td>
<td>Equipment</td>
<td>Amersham Biosiences</td>
<td>Ultraspec 2100 pro</td>
<td>&nbsp;</td>
<tr>
<td>Quartz cuvette</td>
<td>Equipment</td>
<td>Hellma</td>
<td>104-QS</td>
<td>&nbsp;</td>
<tr>
<td>Fast Performance Liquid Chromatography- AKTA purifier 10</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>28-4062-64</td>
<td>&nbsp;</td>
<tr>
<td>Ion exchange column &#x2013; MonoQ 4.6/100 PE</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>10025543</td>
<td>&nbsp;</td>
<tr>
<td>Size exclusion pre-packed column-HiLoad 26/60 Superdex 200</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>17-1071-01</td>
<td>&nbsp;</td>
<tr>
<td>Centricon - Vivaspin15 &#x2013; 10,000 MWCO</td>
<td>Equipment</td>
<td>Sartorius Stedim Biotech GmbH</td>
<td>VS1501</td>
<td>&nbsp;</td>
<tr>
<td>Table centrifuge</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>IEC CL30R</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>Equipment</td>
<td>Bruker Daltonics</td>
<td>Reflex IV</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl (hydrotymethyl) aminomethane</td>
<td>Reagent</td>
<td>BioLab</td>
<td>20092391</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>FRUTROM</td>
<td>235553470</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole</td>
<td>Reagent</td>
<td>Alfa Aesar</td>
<td>288-32-4</td>
<td>&nbsp;</td>
<tr>
<td>EDTA free protease inhibitors cocktail</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-8849</td>
<td>&nbsp;</td>
<tr>
<td>Dnase I (Deoxyribonuclease I)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>DN-25</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Thrombin</td>
<td>Reagent</td>
<td>Fisher BioReagents</td>
<td>BP25432</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>BioLab</td>
<td>07132391</td>
<td>&nbsp;</td>
<tr>
<td>Soudim Dodecyl Sulfate (SDS)</td>
<td>Reagent</td>
<td>BioLab</td>
<td>19822391</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-3148</td>
<td>&nbsp;</td>
<tr>
<td>InstantBlue</td>
<td>Reagent</td>
<td>Expedeon</td>
<td>1SB01L</td>
<td>&nbsp;</td>
<tr>
<td>PageRuler Prestained Protein Ladder</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>SM0671</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification of the m. magneticum strain amb-1 magnetosome associated protein mama&delta;41

磁细菌组成的水生微生物，能够沿着地磁场的方向不同群体。这种行为被认为是帮助他们寻找合适的环境<em&gt;（1）</em&gt;。这种能力是由磁赋予的，一个细胞内的细胞器，包含一个线性链大会脂质囊泡每个能够biomineralize，并附上了〜50纳米磁铁矿或胶黄铁矿晶体。一个原则的功能囊泡的形成，被证明是需要的磁组件是妈妈。妈妈是一个非常丰富的磁相关的蛋白质，这是最特征的磁相关蛋白之一<em&gt;在体内</em&gt;<em&gt;（2-6）</em&gt;。本文的重点，在妈妈的净化，尽管正在研究<em&gt;在体内</em&gt;，没有明确的功能或结构细节已经确定了它。生物信息学分析表明，妈妈是一个四tricopeptide重复（TPR），含蛋白质。 TPR是在广泛的蛋白质折叠的一部分，或形成一个结构基序，它可作为蛋白质相互作用，介导的多蛋白质复合物的模板<em&gt;（7）</em&gt;。 TPRS参与许多重要的任务，在真核细胞的细胞器的进程和许多细菌途径<em&gt;（8-14）。</em&gt;为了理解妈妈，一个独特的TPR，含有蛋白质，高度纯化的蛋白质是必需的第一步。在这篇文章中，我们目前为一个稳定的妈妈缺失突变体的净化（MamAΔ41）协议<em&gt; M。 magneticum</em&gt; AMB - 1。

Watch this Scientific Journal Video about Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÎ”41 at JoVE.com

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

妈妈是一个独特的磁相关蛋白被证明是参与磁激活。在这里，我们从目前的妈妈缺失突变体的净化协议（MamAΔ41）<em&gt; M。 magneticum</em&gt; AMB - 1。

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

purification-m-magneticum-strain-amb-1-magnetosome-associated-protein

Research

JoVE Journal

Biology

13.5K 次观看.  Ben-Gurion University. 妈妈是一个独特的磁相关蛋白被证明是参与磁激活。在这里，我们从目前的妈妈缺失突变体的净化协议（MamAΔ41） M。 magneticum AMB - 1。

视频: Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

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 ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

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 ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.