我们感谢Weixan鲁在文化和维护的Sf9细胞个股的技术援助。 LAY是由医学研究理事会（MRC）研究生奖学金支持。 RJCG是英国皇家学会研究员。结构生物学的牛津司是威康信托人类遗传学中心， 威康信托基金会核心奖授予数量090532/Z/09/Z的一部分。

这个协议假定鼠标kindlin-3基因（FERMT3）已成功地克隆到很晚P10启动子的下游，并且该载体具有侧翼序列的杆状病毒，以允许重组的杆状病毒质粒BAC10 KO一个矢量：1629通过IM琼斯开发和同事28。对于本方案来说，kindlin-3基因被克隆到pOPINE 29和所使用的引物和克隆策略，可以发现在别处12所述。该质粒被工程化以使FERMT3基因（kindlin-3）是P10杆状病毒启动子的控制下与载体包含5&#39;UTR/ORF603和ORF 1629和编码的C-末端His 6 -标签对下游的纯化29。 1。昆虫细胞培养和维护<ol><li>之前的重组杆状病毒的扩增，适于悬浮培养（Sf9细胞） 草地贪夜蛾细胞应生长和维持。 昆虫细胞应该总是使用一个专门的组织培养通风柜无菌技术处理。 </li><li>昆虫细胞的悬浮培养物培养于SF-900 II（SFM），补充有100微克/毫升青霉素和100微克/毫升链霉素的培养瓶中于27℃下用100rpm振荡下的无血清液体培养基中。 </li><li>保持在昆虫细胞培养物的密度范围在1×10 6 - 1个10 7个细胞/ ml通过拆分并用新鲜的SF-900 II介质稀释的细胞培养物。 注意：健康 细胞应该看起来均匀的尺寸，应该是球形的。 </li><li>使用血细胞计数器和光学显微镜来计算培养细胞密度计数细胞中的样品体积。 </li></ol> 2。重组杆状病毒的一代<ol><li>文化与采用SF-900 II介质含100微克/毫升penicilli保持Sf9细胞悬浮n和100微克/毫升链霉素。对于杆状病毒的产生，昆虫细胞，应培养成5×10 5的密度范围- 1×10 6个细胞/ ml。 </li><li>种子约1×10每孔6 Sf9细胞中的无菌的6孔组织培养板中的2ml SF-900 II介质中补充有100微克/毫升青霉素和100微克/毫升链霉素。离开Sf9细胞在RT在通风柜附着于塑料孔的底部，从而形成一个单分子层。 </li><li>通过共转染杆状病毒质粒DNA和质粒DNA到单层培养完成重组杆状病毒的产生。 <ol><li>对于每个转染，混合1-2纯化pOPINE-mFERMT3，其中拥有ORF1629杆状病毒元素，用0.5微克纯化BAC10 KO微克：1629在100微升SF-900 II SFM无抗生素（溶液A）。 </li><li>在一个单独的管，稀释6微升Cellfectin II试剂用100微升SF-900 II SFM无反抗生素对每个转染反应（溶液B）。 A&#39;预混液“可以在这里创建，可降低液体处理，如果许多转染是必需的。 </li><li>混合两种溶液（A和B，约200微升），并在室温下孵育20分钟，以形成脂质-DNA复合物。 </li><li>淡化脂质DNA复合物与800μLSF-900 II SFM无抗生素。小心吸出的Sf9细胞单层介质并小心地吸取溶液A / B和媒体上的Sf9单层的顶端。 </li><li>孵育转染的细胞在湿润的孵化器在27°CO / N和无抗生素的添加进一步1毫升SF-900 II SFM的第二天到每个单层培养。将细胞在27℃延长5天。 </li></ol></li><li>直接从培养液中（约2毫升总数）收获重组杆状病毒并转移到一个干净的离心管中（例如一个15毫升的Falcon管中）。 <ol><li>澄清任何的Sf9细胞DEBR是通过离心在1000×g离心5分钟，在室温。的病毒，这是在所得到的上清液，并标为P1，转移至干净的管中并储存于4℃在黑暗中直到使用。在此阶段，剩余的Sf9单层可用于通过评估重组kindlin-3在昆虫细胞中的存在，评估重组病毒的产生。 </li></ol></li><li>重悬单层用0.5ml PBS中稀释10微升样品与2×SDS-PAGE上样缓冲液等体积​​。加热该样品在&gt; 95℃下进行至少10分钟。 <ol><li>超声处理的样品为1秒，用微型超声波仪末端10％的幅度，如果它太粘适当凝胶上样。 </li></ol></li></ol> 3。重组杆状病毒的扩增<ol><li>对于病毒在悬浮液中的扩增，用1.4×10 6个细胞/ ml的细胞密度，这应占据总烧瓶体积的1/20（ 即 50毫升培养物中一个2升的烧瓶中）。 </li><li>通过与P1病毒原料在感染使用下列公式计算的多个0.1（MOI）感染的昆虫细胞培养物达到的重组病毒的扩增; <img alt="" fo:content-width="4in" src="/files/ftp_upload/51206/51206eq1.jpg" width="400" /> 注：P1病毒产生可以被假定为具有1×10 7 PFU / ml的一个预期病毒滴度。然而，斑块检测可以在此阶段之前进行。 </li><li>孵育在P1感染的昆虫培养在27℃，以100rpm振荡培养3天（72小时）。 </li><li>通过离心分离在1000×g离心5分钟，在室温分离介质将细胞收获病毒。通过评估通过SDS-PAGE和蛋白质印迹kindlin-3蛋白表达保存生成的细胞沉淀在这个阶段进行确认重组病毒的生产。 </li><li>澄清病毒丰富的介质转移到一个干净的试管和储存在4℃黑暗中直到使用。该病毒原料记为P2。 注意：2×10 8 PFU / ml的（或100 PFU /细胞的扩增率）甲病毒滴度可以预期。 </li></ol> 4。 kindlin-3在杆状病毒感染的Sf9表达<ol><li>成长的Sf9细胞培养物悬浮液中的足够量在SF-900 II SFM补充有100微克/毫升青霉素和100微克/毫升链霉素，之前的大规模重组kindlin-3的表达，进行纯化。孵育悬浮培养物在27℃下用100rpm振荡和总培养体积：烧瓶中，以1:5的体积比。 </li><li>感染的Sf9细胞悬浮培养以2×10 6个细胞/ ml的密度。 </li><li>补充的Sf9培养1％的终浓度（V / V）胎牛血清（FBS），随后与扩增重组病毒（P2-病毒原料）给予的1 MOI。孵育感染的培养在27℃，100rpm振荡。 </li><li>嘉实recombina在1000×g离心核苷酸kindlin-3-CHIS 6-表达Sf9细胞72小时后的感染通过离心&#160;并存储所得到的细胞沉淀物于-20℃直至使用，或-80℃下长期贮存。 </li></ol> 5。重组Kindlin-3的纯化<ol><li>解冻冷冻的杆状病毒感染的昆虫细胞（Sf9昆虫）粒料表达重组kindlin-3在冰上。 </li><li>重悬解冻细胞沉淀用裂解缓冲液（50mM的Tris-HCl，pH值7.5，500 mM氯化钠，1％（V / V）吐温20），补充有无EDTA蛋白酶抑制剂混合物和DNase1 1000-2000ü。 注意：可替换地，改性的磷酸盐缓冲盐水（PBS）中也可以使用，其中NaCl的浓度被调节至500mM，以防止内源性的Sf9细胞蛋白并用于下游纯化固定化金属亲和柱（见下文）之间的非特异性相互作用。 </li><li>裂解的细胞通过用洗涤剂和VO孵育悬浮细胞rtexing。声处理（40％振幅，10个循环的10秒脉冲，接着用10秒冷却）的样品在冰浴中进一步破碎细胞或备选地使用Dounce匀浆。 </li><li>澄清离心48,000×g离心1小时，将溶胞产物在4℃下加载得到的上清液到的HisTrap柱（5毫升柱体积）预平衡的裂解缓冲液，在4℃下以1ml /分钟的速率。 注意：可替换地，澄清的裂解物可以用1-5毫升床体积的预平衡的镍琼脂糖珠（ 例如镍琼脂糖凝胶6快速流动）在4℃下1-2小时进行培养。可以通过使用重力流柱的结合步骤之后形成的Ni琼脂糖的柱中。 </li><li>用洗涤缓冲液10倍柱体积的洗涤塔（50毫摩尔Tris-盐酸，pH为7.5，500 mM氯化钠，10mM咪唑），以除去未结合的蛋白。 </li><li>使用AKTA FPLC来洗脱使用从10-500毫米的线性咪唑梯度为10mM /毫升（或在10个柱体积）的速率绑定的重组kindlin-3-CHIS 6。 </li><li>分馏的洗脱到使用AKTA FPLC 0.5-1毫升馏分，用那些含有kindlin-3-CHIS 6通常洗脱，在300 mM的咪唑浓度的馏分。 </li><li>通过SDS-PAGE评估洗脱的蛋白质组合物和首次纯化使用抗His 6抗体或抗小鼠kindlin-3抗体通过Western印迹证实。 </li><li>普尔含有kindlin-3和缓冲液交换成20mM的Tris-盐酸，pH为7.5，200 mM氯化钠通过一系列的稀释液为缓冲液和样品的浓度使用离心浓缩蛋白具有50 kDa的分子量截止（MWCO）的级分在4℃下 注：此外，使用滑入式A-仪透析盒为30 kDa的截留分子量透析的蛋白质溶液在4℃4小时，以O / N为20毫米的Tris-HCl，pH值为7.5，200 mM氯化钠。 注意：对于离子交换（见下文）的NaCl的浓度较低， <e米&gt;即50毫米，可与已被用于此步骤。 </li><li>应用缓冲交换蛋白溶液涂布使用AKTA FPLC以0.5ml /分钟的速度预平衡的HiTrap肝素HP柱（5毫升柱体积）。 注意：绑定kindlin-3中相同的缓冲液用线性NaCl梯度（0.2 M氯化钠至1M NaCl）中，于10毫/毫升的速率增加洗脱。 Kindlin-3-CHIS6有望洗脱在〜0.6 M氯化钠。 </li><li>分馏的洗脱入0.5-1毫升的级分，并在适当时评估通过SDS-PAGE和免疫印迹的蛋白质组合物，。 注意：人们可以期望的接近95％的蛋白质纯度，如评估通过SDS-PAGE。 </li><li>池馏分含有kindlin-3和集中使用离心机浓缩蛋白具有50kDa的截留分子量，以0.5-2毫升的最终体积。 </li><li>在最后的步骤中，研磨的浓缩的蛋白质和缓冲用尺寸排阻色谱法（SEC）交换蛋白。 <ol><li>应用纯化蛋白到苏perdex S200（16/60）或（10/30），以1毫升/分钟或0.5ml /分钟取决于柱尺寸的比率预平衡在20mM的Tris-HCl，pH值7.5，200 mM氯化钠，1mM DTT的使用。 注：在磷酸缓冲盐水（PBS）中，如果需要的尺寸排阻色谱法，也可以进行。 </li><li>根据大小通过将缓冲在柱上以1毫升/分钟或0.5ml /分钟，这取决于所用的列大小的速率纯化蛋白质。 注：蛋白质从柱上洗脱分馏和监视用280nm处的吸光度。 </li><li>一个单一的吸收峰应该从SEC，这是分馏并通过SDS-PAGE进行评估，以确定均匀性，通常是&gt; 95％纯后此步骤可以预期的。 </li></ol></li><li>浓缩纯化kindlin-3-CHIS 6用离心机浓缩蛋白具有50kDa的截留分子量，以〜15毫克/毫升，作为使用的109320 M A计算的消光系数（ε）评估分光光度计-1厘米-1 &lt;/ SUP&gt;（假设所有半胱氨酸残基被还原）。 </li><li>对于存储在-20℃下和长期贮存于-80℃，等分试样的蛋白质进入PCR管和闪蒸冷冻在液氮中的样品。或者，该蛋白可直接使用多种生物化学和生物物理技术用于调查。 </li></ol>

<ol>
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作者什么都没有透露。

杆状病毒表达系统正变得越来越流行，并用于生产重组蛋白的毫克量的用于使用生物物理研究，包括X-射线晶体蛋白质表征的重要工具。尽管是更苛刻的实验杆状病毒表达系统提供了几个优点E。大肠杆菌其中之一是用于真核来源，例如适当的分子伴侣和翻译后修饰的机会存在的蛋白质的近天然环境中。在我们自己的努力来表达kindlin-3，另类表达宿主中使用，包括哺乳动物细胞系和细菌表达菌株（未发表意见）。一般地，许多大肠杆菌大肠杆菌菌株测试产生了非常少量的重组kindlin-3（〜0.5 mg / L的文化;未发表意见）。然而，在昆虫细胞的杆状病毒驱动的表达是特别有效的，并在共同mparison到哺乳动物细胞中，更适合于产生所需的隔离一毫克的重组胞质蛋白（未发表的观察结果）的大生物量的瞬时表达。我们推测，存在真核生物伴侣可能会允许高效率的生产kindlin-3。 在杆状病毒感染的昆虫细胞和重组蛋白表达在这项工作中使用的杆状病毒是基于苜蓿银纹夜蛾核型多角体病毒（的AcNPV）。自然界中的AcNPV，传染的苜蓿银纹夜蛾 （苜蓿修剪器）昆虫幼虫，要求多角体蛋白以形成闭塞由此病毒体被封装在一个结晶蛋白基质从而为他们的释放必要的保护。在培养的细胞中是不需要闭塞体的形成进行复制，因此是可有可无的。在表达外源蛋白质的情况下的多角体蛋白基因可以是Replaced在重组的AcNPV的基因对感兴趣的蛋白质。可以的AcNPV感染其他鳞翅目昆虫的物种和重组蛋白表达粘虫草地贪夜蛾的目的蛹卵巢细胞使用。在此处所述的方法中，杆粒的AcNPV（BAC10）被设计以使得必需病毒基因，ORF1629，是由氯霉素乙酰转移酶产生的敲除杆状病毒质粒（BAC10：KO 1629）的插入失活，使得其无法形成传染性baculovirions 28。共转染Sf9细胞用线性化BAC10的：KO 1629和含FERMT3转移载体修理无效ORF1629，通过转置，产生一个可行的基因组中还掺入了多角体蛋白启动子28的控制下，FERMT3基因。 我们通过一个THRE描述纯化方案高纯度重组小鼠的隔离kindlin-3Ë步色谱方法。这里所用的方法可以很容易地应用到其他的His-标记的蛋白。我们采用的离子交换步骤，以进一步纯化kindlin-3，但是我们相信，这是又一个伪亲和步骤作为kindlin-3拥有大量的碱性残基，包括其内的F1域多聚赖氨酸伸展。此外，kindlin-3被认为是结合并与质膜，在那里它的功能的胞质面相互作用，因此我们预测，碱性残基的聚类将使蛋白质，以对抗负电荷的膜。 在纯化方案中描述的缓冲器被认为是标准的和常用于结构上的生物。该thermofluor测定（ 图5）表明，kindlin-3是稳定的，在大多数的缓冲条件在pH值高于6.0。 β1：学习kinldin-3时，这是特别有用的和重要的，通知我们的实验尾部相互作用的NMR，取得了优异的光谱，在pH 6.1的低浓度的NaCl 12。 之前任何生物物理研究可以进行，以证明所关注的纯化蛋白确实是正确折叠的并且是功能活性是重要的。在以前的出版物中，我们证明了重组kindlin-3表达和纯化使用这种方法是一种单体和单分散在溶液中，所评估的尺寸排阻色谱法，动态光散射，分析超速离心法和小角度X射线散射，和是还能够结合并认识到膜远侧NPxY和β1A胞质尾区14上游的丝氨酸/苏氨酸簇，从而确认它的行为类似于天然蛋白，这是与以前的细胞和生理研究14,20,22线。使用热稳定性试验的建议是个额外的方法目的蛋白质电子正确折叠，如不正确折叠的蛋白质，将导致高荧光背景由于暴露的疏水残基。 蛋白质的kindlin家庭，因为他们在体内的整合至关重要共激活因子意想不到的作用被发现一直是备受瞩目的焦点。这引发了很大的力气，重组表达出来并解决它们的结构。迄今为止有限的成功已报道在表达全长重组蛋白毫克量，但我们在这里描述的使用杆状病毒系统，它允许在水平，其中结构的研究变得可行大规模的表达。通过产生大量重组kindlin-3，我们可以预计，这将有助于该蛋白质的进一步研究。这里描述的重组鼠kindlin-3杆状病毒驱动的方法和纯化工作流也可以用于表达和纯化的其他kindlin亚型，这也是难以表达，并具有多聚赖氨酸延伸，并且可以被进一步适配为其他胞质蛋白，如核酸结合蛋白，该失败的细菌菌株来表达。

该kindlin家族蛋白是粘着组件的重要组成部分，因此对复杂的生命至关重要。 Kindlins，其中有3个亚型在哺乳动物（kindlin-1，kindlin-2，和kindlin-3），被认为是细胞外受体整合大林一起共激活因子1。整合素介导的细胞粘附的细胞表面在高等真核生物连接到细胞外基质（ECM）。它是在过多的生理现象，它包括组织的完整性，胚胎，骨代谢，止血，和免疫力的一个重要而常见的过程。整合素介导的细胞黏附是通过由内到外信号传导途经塔林和kindlin到整合的β-亚单位胞质尾（CTS）在他们的保守NPxY图案的结合激活。然而kindlin蛋白质的生物医学重要性一直延伸到细胞核，在那里kindlin-2已经显示出在最近一些报告是参与转录人控制2,3。 Kindlins是约75 kDa的多结构域蛋白，由一个二分C-末端FERM（4.1带，埃兹蛋白，根蛋白，膜突蛋白）结构域，它是由一个PH结构在其F2的子域的中心（PH）结构域中断的占有纯度标记4,5。该kindlin-2和kindlin-3 PH结构域的研究表明，它结合到脂质第二信使磷脂酰肌醇（3,4,5） -三磷酸肌醇和磷脂酰肌醇-（4,5） -二磷酸6-8。然而，kindlin-1 PH结构域的研究表明，它结合到磷脂酰肌醇（3,4,5）P 3具有低得多的亲和性，可以在kindlin-1同种型特异的盐桥防止脂质的结合来解释9。此外，有插入到被预测为被展开，但结合到磷脂酰丝氨酸在质膜10,11的内小叶的kindlins的F1域的约100个氨基酸的循环。该kindlin FERM域被认为是同源的塔林FERM区域，虽然大林FERM域不具备PH结构域。既kindlins和大林与对整合的β-尾巴NPxY图案通过他们的FERM域的F3区域互动，但kindlin结合膜远端主题，而大林针对近膜1 12-16。 Kindlins和踝蛋白二者除了具有与未在其他FERM蛋白11,17发现了泛蛋白-样折叠的N-末端结构域F0。已经研究kindlin-2的基频域表明，它独立地结合至磷脂酰肌醇（4,5） -二磷酸富集膜17。 该kindlins表现出旁系同源基​​因特异性组织表达模式和非冗余的生理功能。 Kindlin-1主要表达在表皮，而且在较小的程度结肠，胃，肾; kindlin-2广泛表达，但主要集中在横纹肌和平滑肌肉是表达在胚胎发育的4只kindlin以及kindlin-3的表达与kindlin-3的最高浓度在18巨核细胞中发现的造血组织。然而，最近的研究表明，功能蛋白表达在内皮组织以及19。 Kindlin-3是急性医疗利益由于在血液中其重要的生理作用。它是血小板聚集和血栓中形成20，白细胞滚动响应于感染和炎症21,22和破骨细胞的形成足细胞骨吸收23扩散的关键。此外，kindlin-3在人体内的消耗导致白细胞黏附缺陷症III型-一个疾病的特点是威胁生命的出血性疾病及复发性细菌感染20,24,25。 Kindlin-3基因敲除小鼠的研究表明在该蛋白质的关键功能细胞粘附KIND3 - / -小鼠由于不活跃的血小板整合素，严重的骨硬化病，以及受损的白细胞粘附20,22显示不同的表型，如严重出血，类似症状的人缺乏kindlin-3。 在kindlins高分辨率结构数据，迄今为止，已经被限制到单独的子域，如PH结构的kindlin-1 9和kindlin-2 26,27（PH）结构域和kindlin-1 11和kindlin的基频域-2月17日 。最每个kindlin多肽的子域都不过抵制克隆和结构分析（耶茨和吉尔伯特，未发表的观察结果）和全长蛋白的研究已经阻碍了通过使用大肠杆菌表达和纯化足够量的难度大肠杆菌 （未出版的观察和阿尔布热等人 14）。有一个在kindlin-3及其夫相当的医疗利益nction，旁边的另外两名家庭成员，最近我们产生它的毫克量由重组蛋白表达的杆状病毒感染12驱动草地夜蛾细胞。因此，我们在这里描述了用于生产重组小鼠kindlin-3在昆虫细胞培养毫克量，适合于广泛的结构研究和生物化学分析的方法。 在这个协议中，我们使用一个精心设计的淘汰赛杆粒（BAC10 KO：1629）的 也就是说，孤独，无法产生可行的病毒体28。因此，病毒DNA被救出重组与转移载体，在这种情况下，还包括kindlin-3基因（FERMT3）和结果的FERMT3基因取代病毒非常晚期基因，这是高度表达的，但冗余，导致重组病毒表达小鼠kindlin-3作为病毒的生命周期28的一部分。 我们确定了这个方法用于生产kindlin-3后尝试在其他表达宿主表达和纯化事实证明比登天还难（未发表意见），而且由于人口信息网矢量套件，这是我们用于克隆，而且可以在许多表达部署的多功能性主持29。

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			<td height="21" style="height:21px;width:467px;">Sf-900 II serum free media (SFM) 1X liquid</td>
			<td style="width:123px;">Life Technoligies</td>
			<td style="width:119px;">10902-096</td>
			<td style="width:439px;">store at 4&#176;C and warm to room temperature before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">Cellfectin II Reagent</td>
			<td style="width:123px;">InVitrogen</td>
			<td>10362-100</td>
			<td>Alternatively, GeneJuice transfection (EMD) reagent can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">Streptomycin Sulphate (solid)</td>
			<td style="width:123px;">Melford</td>
			<td>S0148</td>
			<td>Sterilise filter (0.22&#956;m filter) before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">Penicillin G, potassium salt (solid)</td>
			<td style="width:123px;">Melford</td>
			<td>P0580</td>
			<td>Sterilise filter (0.22&#956;m filter) before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">CELLSTAR Sterile 6-well Culture Plate</td>
			<td style="width:123px;">Greiner bio-one</td>
			<td style="width:119px;">657160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:123px;">Life Technologies</td>
			<td>10100-147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">Protease Inhibitor Cocktail</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">P8849</td>
			<td>Caution: Protease inhibitors are dissolved in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">Bovine pancrease deoxyribonuclease (Dnase) I&#160;</td>
			<td style="width:123px;">Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">HisTrap FF (5ml)&#160;</td>
			<td style="width:123px;">GE Heathcare</td>
			<td>17-5286-01</td>
			<td>Requires an &#196;kta FPLC machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">HiTrap Heparin (5ml)</td>
			<td style="width:123px;">GE Heathcare</td>
			<td>17-0407-01</td>
			<td>Requires an &#196;kta FPLC machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:467px;">Amicon Ultra-15 Centrifugal Filter Units (with Ultracel-50 membrane)</td>
			<td style="width:123px;">Millipore</td>
			<td>UFC905024</td>
			<td style="width:439px;">15ml capacity and a MWCO of 50kDa protein concentrator</td>
		</tr>
	</tbody>
</table>

efficient production and purification of recombinant murine kindlin-3 from insect cells for biophysical studies

Kindlins是必不可少的辅激活，与踝蛋白，细胞表面受体的整合素和也参与整合外到内的信令，并在细胞核中基因转录的控制。该kindlins是〜75 kDa的多结构域蛋白质和绑定到NPxY主题和整合素β亚基胞质尾上游T / S簇。在血栓形成的hematopoietically重要kindlin亚型，kindlin-3，是至关重要的血小板聚集，白细胞滚动对感染的反应与炎症和破骨细胞的足细胞形成骨吸收。 Kindlin-3在这些进程中的作用，导致大量的细胞和生理研究。然而，有必要获取蛋白质的高品质毫克量进一步研究的有效方法。我们通过使用杆状病毒-D的开发的协议，这里描述的，用于重组鼠kindlin-3的高效表达和纯化四分五裂表达系统在Sf9细胞中产生足够量的高纯度的全长蛋白质，以允许它的生物物理表征。同样的方法可以采取在其他哺乳动物kindlin亚型的研究。

重组小鼠kindlin-3采用杆状病毒感染的Sf9细胞的大规模表达可以不到两周的时间来实现毫克级，如图所示在原理图如图1A，只需要少量的质粒DNA从一个QIAprep小量制备试剂盒，用于例子。重组杆状病毒的产生是通过共转染Sf9细胞与kindlin-3（FERMT3）含有质粒连同一个精心设计的线性化的杆状病毒质粒（BAC10：KO 1629）鼠标实现后5-7天收获新形成的病毒颗粒， 如图所示1A。的杆状病毒产生的结果在100％的重组病毒的这种方法，基本上省去需要进行噬斑纯化28,30。有代表性的小规模（2毫升单层）培养将产生每毫升的重组含病毒液以1×10 7噬斑形成单位的预期病毒滴度（pfu的）文化。一个可以执行斑块检测来确定实际的病毒滴度，但是这也许是过于劳动密集时，大量的构造进行测试结构研究。病毒生成步骤的成功可以通过使用一个含有EGFP-质粒并行进行评估，或者对于这个kindlin-3构建体，该染Sf9单层可重新悬浮在PBS中，通过SDS-PAGE和蛋白质印迹评估，这通常表明一个明确的频带相对应的75 kDa的His-标记的蛋白， 如图1B所示 。病毒随后被放大，以产生足够量的用于大规模（升体积）昆虫细胞感染和重组蛋白的隔离。第二通道病毒（P2）是通过感染悬浮培养物用0.1的MOI估计（见协议）生成的。至关重要的是，放大的Sf9悬浮培养占总瓶量只有二十分之一。这个附加的曝气确保生成的vir区含有我们的媒体，后3天（72小时）感染后收获，将产生在随后的表达培养足够数量的kindlin-3。扩增的病毒原液（P2）被假定为具有2×10 8 PFU / ml的基于100 PFU /细胞的使用/毫升在扩增31的2×10 6个细胞的细胞密度的保守估计，预计的病毒滴度。通常，为了获得最佳的杆状病毒的扩增和蛋白质生产，Sf9细胞应是均匀的尺寸和球形的， 如图1C所示 。此外，病毒的扩增和蛋白表达为最大在72小时后感染，无论是在96小时后的感染是显著降低。 一旦病毒已被放大，并用于感染Sf9细胞在大规模实验重组kindlin-3被部分地由于其设计的C-末端His 6 -标签的使用固定化金属亲和色谱的纯化matography，如示于图2。它来进行纯化在4℃下是很重要的，并有足够的蛋白酶抑制剂已被添加，以防止蛋白水解。该部分纯化的kindlin-3进一步纯化，以通过离子交换色谱（IEC）使用肝素柱附近的均匀性， 如图3。我们采用代替常规的离子交换柱，用肝素柱的，如我们预测的大量碱性氨基酸残基，包括kindlin-3 F1域内的多聚赖氨酸的伸展，将与带负电荷的硫酸酯基团的强相互作用之列。这种策略是用于DNA和RNA结合蛋白与碱性核酸结合的补丁是特别有用的，例如uridylyltransferase CID1 32的RNA结合终端。最后kindlin-3纯度是“打磨”由尺寸排阻色谱法，以除去团粒，达到同质性，如图图4。在该协议中指定的缓冲区是常用于蛋白质纯化用于结构分析的标准缓冲液。通常，避免了基于磷酸盐的缓冲液对纯化蛋白质的结构研究，特别是结晶筛选由于磷酸盐结晶中的结晶粒（特别是用于在4℃下的实验）的形成。然而，我们进行了Thermofluor基于热位移分析，以确定哪些缓冲液稳定为kindlin-3， 如图5。简言之，将纯化的蛋白质溶液稀释到缓冲液覆盖的范围的pH值和氯化钠浓度，因此形成一个2维的画面。蛋白质的熔化是通过从SYPRO橙染料（分子探针），其结合到所述折叠蛋白核心内的疏水残基，在温度范围20-95℃（293-368 K）观察荧光测定。温度中间点时THE蛋白的展开使用的Opticon Monitor软件 （转变温度，T M）计算并在别处33描述。 Kindlin-3，观察到的pH值范围为7.0-9.0范围内是稳定的高的氯化钠浓度（500毫米），具有一致化转变温度（T M） 55°C。也有人指出，Kindlin-3的Tm值约55℃的pH值范围7.0〜7.5范围内，不论氯化钠浓度。 我们发现，有kindlin-3的限制性蛋白水解的纯化过程中，但高度浓缩的蛋白质（〜15毫克/毫升）的SDS-PAGE分析表明有限污染相等强度的两个额外的多肽。奇怪的是，但是有一个由尺寸排阻色谱法没有显示额外的物种，如示于图4。因此，它被认为是该蛋白被蛋白酶缺口但仍然折叠和hydrodyn从全长蛋白amically没有什么区别。有趣的是，钙蛋白酶是已知的蛋白酶裂解kindlin-3在Tyrosine373，这是在普列克底物蛋白同源（PH）结构域34的β1-β2循环，并且可以解释我们的观测。此外，蛋白质印迹显示，该多肽组成的双重峰1具有C-末端His-标签和双峰的表观分子量，总结时，等于75 kDa的分子量相同的天然蛋白质。纯化的重组kindlin-3每升Sf9细胞（〜2个G细胞重量）的产量，充其量，5毫克。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig1highres.jpg" src="/files/ftp_upload/51206/51206fig1.jpg" /> 图1。杆状病毒generatio的杆状病毒感染的昆虫细胞异源蛋白表达的概述。（A）系统化概述在Sf9细胞n和kindlin-3的表达。在DNA载体的原理图，5&#39;UTR / ORF603被着色为红色，则ORF1629被着色为绿色，并且的兴趣点（POI）的蛋白质的基因为蓝色。 （B）中 免疫印迹的6小规模（2毫升单层）培养的Sf9细胞制备重组杆状病毒和重组鼠kindlin-3（泳道根据培养标记），使用抗His 6抗体。 （C） 在SF-900 II SFM中悬浮​​生长健康Sf9细胞的光学显微镜图像补充有抗生素，并转移到35毫米的孔组织培养皿中（见协议）。图片是20X的放大倍率。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig2highres.jpg" src="/files/ftp_upload/51206/51206fig2.jpg" /> 图2。Represekindlin-3通过固定化金属亲和层析（IMAC）ntative纯化，SDS-PAGE镍亲和纯化的重组鼠kindlin-3感染的Sf9细胞（〜2克细胞重量）杆状病毒表达。使用咪唑梯​​度（凝胶如上所示）吸附的蛋白质洗脱。车道标记如下; MW，分子量标记;赖氨酸，全细胞裂解物;金融时报，流过（未绑定），威斯康星，洗1次，WII，洗净2。还进行Western blot分析（下）使用洗脱组分，以确认在重组蛋白的工程His标签的存在。 <a href="https://www.jove.com/files/ftp_upload/51206/51206fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig3highres.jpg" src="/files/ftp_upload/51206/51206fig3.jpg" /> 图3。 REPRE在280nm（蓝色）显示单一对称峰下使用的0.05-1.0 M的NaCl浓度范围内的线性氯化钠梯度（绿色）洗脱（kindlin-3通过肝素亲和层析的纯化sentative（A）洗脱曲线观察B）的SDS-PAGE分次洗脱展示75 kDa蛋白的存在，分析，kindlin-3（标K3）。 <a href="https://www.jove.com/files/ftp_upload/51206/51206fig3highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig4highres.jpg" src="/files/ftp_upload/51206/51206fig4.jpg" /> 图4代表凝胶过滤色谱法纯化 kindlin-3的浓kindlin-3（A）的凝胶过滤洗脱图用的Superdex S200（16/60）在Tris-HCl，pH值7.5,150 mM氯化钠和1mM DTT在20℃下根据洗脱体积上，kindlin-3迁移如预期的75 kDa的蛋白，这表明它主要是单体的。 （B）的SDS-PAGE的高浓缩纯化的重组kindlin-3为14.5毫克/毫升。 <a href="https://www.jove.com/files/ftp_upload/51206/51206fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig5highres.jpg" src="/files/ftp_upload/51206/51206fig5.jpg" /> 图5。Thermofluor基于热位移检测方法的缓冲液筛选。Kindlin-3稀释成含有pH值对氯化钠浓度的二维屏幕上的各种缓冲液。化转变温度（温度中点）通过荧光观察疏水作用结合染料，SYPRO橙（分子探针）和使用的Opticon Monitor软件来计算。 （ 一 ）转变温度的3D直方图与（B）从50.4°C。计算出的平均值在转变温度的变化一起绘制为了清楚起见酒吧是根据温度，以其所对应的范围着色。 <a href="https://www.jove.com/files/ftp_upload/51206/51206fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies at JoVE.com

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins是基本的细胞粘附通过整合，但他们的研究已经阻碍了在细菌宿主中重组表达他们所遇到的困难。我们在这里描述它们高效的生产在杆状病毒感染的昆虫细胞的方法。

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

efficient-production-purification-recombinant-murine-kindlin-3-from

Research

JoVE Journal

Biology

23.3K 次观看.  University of Oxford.  Kindlins是基本的细胞粘附通过整合，但他们的研究已经阻碍了在细菌宿主中重组表达他们所遇到的困难。我们在这里描述它们高效的生产在杆状病毒感染的昆虫细胞的方法。 

视频: Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

科研

生物学

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

细菌，昆虫细胞和植物系统：重组蛋白表达在不同的生物工厂的比较分析

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.