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。

<table><tbody><tr><td>Sf-900 II serum free media (SFM) 1x liquid</td><td>Life Technologies</td><td>10902-096</td><td>store at 4 &amp;deg;C and warm to RT before use</td></tr><tr><td>Cellfectin II Reagent</td><td>Invitrogen</td><td>10362-100</td><td>Alternatively, GeneJuice transfection (EMD) reagent can be used</td></tr><tr><td>Streptomycin sulphate (solid)</td><td>Melford</td><td>S0148</td><td>Sterilize filter (0.22 &amp;mu;m filter) before use</td></tr><tr><td>Penicillin G, potassium salt (solid)</td><td>Melford</td><td>P0580</td><td>Sterilize filter (0.22 &amp;mu;m filter) before use</td></tr><tr><td>CELLSTAR Sterile 6-well Culture Plate</td><td>Greiner Bio-One</td><td>657160</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>10100-147</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>P8849</td><td>Caution: Protease inhibitors are dissolved in DMSO</td></tr><tr><td>Bovine pancrease deoxyribonuclease (Dnase) I&amp;nbsp;</td><td>Sigma</td><td>D5025</td><td></td></tr><tr><td>HisTrap FF (5 ml)</td><td>GE Heathcare</td><td>17-5286-01</td><td>Requires an &amp;Auml;kta FPLC machine</td></tr><tr><td>HiTrap Heparin (5 ml)</td><td>GE Heathcare</td><td>17-0407-01</td><td>Requires an &amp;Auml;kta FPLC machine</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units (with Ultracel-50 membrane)</td><td>Millipore</td><td>UFC905024</td><td>15 ml capacity and a MWCO of 50 kDa 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

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

蛋白晶体的微结晶和<em&gt;在Cellulo</em&gt;衍射

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

科研

生物化学

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 ...

生物学

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

改进杆状病毒表达载体生产昆虫细胞分泌植物蛋白

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

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, ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

免疫与感染

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

用于神经元细胞贩运研究的管子中纯化和直接单生物素重组BDNF的改进协议

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

神经科学

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

重组蛋白表达的结构生物学在HEK 293F悬浮细胞:一种新的和可访问方法

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

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

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蛋白晶体的微结晶和