This work was funded by a grant from the National Institutes of Health (R15 AI117670) to B.M.S and P.J.W, as well as a Cottrell College Scholar Award from the Research Corporation (20185) to P.J.W. L.M.M. was supported by a Provost's Fellowship from CMU.

1.十大大肠杆菌表达TRET纯化注意:请联系作者，要求TRET表达大肠杆菌 （的pBAD质粒TRET，含蛋白质阿糖胞苷的控制，转化E.十大下T. TENAX TRET基因大肠杆菌 19）及所附材料转让协议。以下方案通常给出的约4毫克/升的蛋白质产量。 <ol><li>准备TRET表达大肠杆菌 3毫升过夜培养。 <ol><li>条纹十大大肠杆菌与一个LB培养基（LB）含有100μg/ mL氨苄青霉素的琼脂平板的pBAD-TRET表达载体转化。 </li><li>孵育所述板在37℃下约48小时。 </li><li>从板挑取单菌落，接种3毫升含在培养管100微克/毫升氨苄青霉素的LB液体培养基中。 </li><li>放置在振荡培养箱所述管在37＃176;Ç点¯x175转过夜。 </li></ol></li><li> 诱导TRET表达大肠杆菌蛋白的表达。 <ol><li>添加750毫升了不起肉汤补充有100微克/毫升氨苄青霉素到2800毫升费恩巴赫培养瓶中。转让1毫升肉汤从烧瓶到试管为作为空白以后使用。 </li><li>添加在步骤1.1.4至培养烧瓶中产生的3毫升过夜培养，然后将烧瓶在培养箱并在37℃×200转动摇。定期检查文化的吸收在600纳米与步骤1.2.1收集的空白。 </li><li>一旦在600的吸光度0.5-1.0之间处达到，通过加入1M的阿拉伯糖溶液（终浓度为1mM），以在培养750微升诱导TRET表达。烧瓶返回到培养箱中，在37℃下×200转摇过夜。 </li></ol></li><li> 颗粒和裂解TRET表达大肠杆菌细胞。 <ol><li>转移文化聚丙烯bottle和离心机在4000 xg离心在4℃下15分钟。 </li><li>弃去上清液并重新悬浮在15ml磷酸盐缓冲盐水的（PBS）中沉淀。 </li><li>在4000 xg离心细胞悬液转移至50 mL锥形管并离心15分钟，4℃。弃去上清液并继续到细胞裂解（步骤1.3.4）或在-80℃长期保存沉淀。 </li><li>在50mL锥形管溶解1蛋白酶抑制剂的迷你片剂在20ml洗涤缓冲液（50mM的NaH 2 PO 4，500 mM氯化钠，的20mM咪唑，pH 8.0）中的。 </li><li>蛋白酶含抑制剂的洗涤缓冲液转移到含颗粒的锥形管中。涡旋直至沉淀重新悬浮。 </li><li>传送再悬浮细胞至一个100毫升烧杯中，并通过超声处理裂解细胞（上45秒脉冲序列45秒断用2分钟和15秒的运行时间以75％的幅度）。 </li><li>转移裂解物至50 mL金属锥形管和离心机在15000 xg离心在4℃下60分钟。 </li><li>通过0.2-0.45微米注射器过滤到50mL锥形管澄清通道的裂解液。 注意:得到的溶胞产物的典型浓度为100毫克/毫升。 </li></ol></li><li> 用快速蛋白液相层析（FPLC）从大肠杆菌细胞裂解物纯化TRET。 <ol><li>设置了FPLC与镍亲和柱（5毫升床体积）。用10毫升去离子水，或直到柱是清洁的污染物洗柱。平衡，用20毫升的洗涤缓冲液的柱（50毫米的NaH 2 PO 4，500 mM氯化钠，的20mM咪唑，pH 8.0）中以1毫升/分钟的流速。 </li><li>装载从步骤1.3.8获得至色谱柱上的裂解液（20毫升）中，在1毫升/分钟的流速洗脱用洗涤缓冲液未加标签的蛋白质，直到吸光度达到背景水平（通常80-100毫升的洗涤缓冲液是必需的） 。 </li><li>洗脱His-标记TRET通过使用线性克从1-100％在60分钟内以1毫升/分钟的流速洗脱缓冲液（50mM的NaH 2 PO 4，500 mM氯化钠，250mM咪唑，pH 8.0）中的radient。收集4毫升馏分直到TRET已洗脱的吸光度达到基线水平。 注意:通常情况下，需要60毫升洗脱缓冲液以洗脱蛋白质，并且该蛋白质在60％-100％的洗脱缓冲液范围洗脱。大约10-15毫升纯TRET在洗脱缓冲液中获得。 </li><li>通过在针对洗脱缓冲液空白测量280nm处的吸光度确定TRET的浓度。 </li></ol></li><li> 交换TRET成三（羟甲基）氨基甲烷（TRIS）通过透析缓冲液中。 <ol><li>根据制造商的说明制备透析管后，通过用离子交换水，然后的Tris缓冲液（50mM的Tris，300mM的氯化钠，pH值8.0）冲洗素它。 </li><li>加载TRET样品放入使用注射器和钝针透析管。透析过夜抗拒着2升Tris缓冲。 </li><li>通过在对从透析洗涤收集的空白测量280nm处的吸光度确定TRET的浓度。 </li><li>转移TRET溶液到50毫升锥形管中，并进行到海藻糖类似物合成（步骤2）或酶储存于4℃。 注:TRET是一个热稳定的蛋白。该TRET在4℃下保存在Tris缓冲液中数月而不观察活动的显著损失。 </li></ol></li></ol> 2.使用TRET海藻糖酶类似物的一步合成注意:下面的协议描述了基于4毫升容积的反应规模，可以根据反应的效率和产品的分子量提供约15-30毫克海藻糖类似物。反应组分可以缩放如果需要的话，以获得更多或更少的海藻糖类似物。 <ol><li>添加葡萄糖类似物（0.080毫摩尔，质量将取决于分子量），UDP-葡萄糖（0。160毫摩尔，97.6毫克）， 氯化镁 （0.080毫摩尔，16.3毫克）于15毫升锥形管中。这些组分的最终浓度将是分别为20毫米，40毫米和20毫米。 </li><li>添加TRET在Tris缓冲液（来自步骤1.5.4获得），如果有必要，Tris缓冲液的适当体积（50毫摩尔Tris，300mM的氯化钠，pH值8.0），以达到300微克/毫升和最终的最终酶浓度的4mL的体积。吸管将混合物上下轻轻或反转管以溶解固体。 </li><li>孵育在70℃以300rpm振荡1小时的反应，然后置于冰上的管冷却。 </li></ol>从原油酶促反应混合物海藻糖类似物3.净化<ol><li>直到所有的液体通过过滤器的预冲洗离心过滤单元（标称分子量限度（NWML）10 kDa）的加入3毫升去离子水至离心过滤器单元，并在3000 xg离心离心以除去痕量甘油在膜入管（大约20分钟）。重复两次。完成之前立即或在反应（步骤2.3）在这一步。 </li><li>冷却该酶促反应混合物（来自步骤2.3获得）后，将其转移到预漂洗离心过滤单元。冲洗反应管用1mL的去离子水，并转移到离心过滤单元。对于产品的最大回收反应管重复漂洗。 </li><li>在3000 xg离心离心离心过滤单元，直到所有的液体通过过滤器进入管（大约20分钟）。在3000 xg离心冲洗离心过滤单元与3毫升去离子水和离心机上腔室，直到所有的液体通过过滤器进入管（大约20分钟）。重复漂洗产品的最大回收。 </li><li>丢弃离心过滤器单元的上部腔室。混合床离子交换树脂（3克）到滤液中添加在管（典型滤液体积的底部存储介质为8-15毫升取决于冲洗的数目）。在室温下搅拌1小时，以足够能保持悬浮在溶液中的树脂珠粒的速度一磁力搅拌棒。 </li><li>倒出上清液并将其过滤以除去树脂。加入5毫升的去离子水来冲洗剩余的树脂。倒出上清液并过滤，它与从第一滗析的产物溶液合并。树脂产品的最大回收重复清洗。 </li><li>分析薄层色谱（TLC）或高效液相色谱法的反应来确定葡萄糖类似物的原料的海藻糖类似物产物的完全转化是否达到了。参见步骤4.1 TLC分析和步骤4.2进行HPLC分析。 </li><li>通过冻干或旋转蒸发除去水，得到干燥产物。如果TLC或HPLC分析期间没有观察到未反应的葡萄糖类似物，纯化通过色谱法是不必要的。权衡产物，得到的反应一愕LD和执行核磁共振（NMR）光谱分析（步骤4.3），以确认产品的结构和纯度。 </li><li>如果TLC分析过程中观察到的未反应的葡萄糖类似物，它从用尺寸排阻柱海藻糖类似物分开。 <ol><li>准备根据制造商的说明含有去离子水饱和的，超细的P2聚丙烯酰胺珠大小排阻介质1×100厘米柱。 注:尺寸排阻柱可以用离子交换水洗涤后可重复使用。 </li><li>重新溶解的干燥酶反应产物在0.5毫升去离子水（来自步骤3.7获得）。应用该产物溶液，以手动的大小排阻柱或通过使用柱流速适配器。冲洗包含具有去离子水另一个0.5mL的粗产物小瓶，并且将其装载到尺寸排阻柱。 </li><li>通过重力流洗脱用去离子水产物和收集的2μm左右馏分1L体积。 </li><li>通过TLC（步骤4.1）分析级分。汇集含纯海藻糖类似物的级分。 </li><li>通过冻干或旋转蒸发除去水，得到干燥产物。权衡产物，得到反应产率，并继续NMR分析（见步骤4.3）。 </li></ol></li></ol> 4.海藻糖模拟产品分析<ol><li> 执行TRET反应薄层色谱（TLC）分析。 注意:此过程也可以用来分析大小排阻柱级分。可能有必要集中之前TLC分析，以在TLC板上观察化合物染色反应混合物或柱级分。 <ol><li>标记泳道用铅笔TLC板表面上，并应用分析物和相关标准（多个）到适当的车道，包括葡萄糖类似物标准，海藻糖类似物标准（如果有的话），将反应混合物（或馏分从收集规格Exclusion柱纯化），和共点。施加各样品的TLC板后，让板干燥。 注:对于反应分析，样品通常为2微升加到TLC板。 </li><li>开发使用正丁醇/乙醇/去离子水的TLC板（5:3:2）。 </li><li>擦干开发薄层板上，然后将它浸入5％H 2 SO 4的乙醇（糖染色），热上高设置一个热板，直到含糖点可以可视化（一般为5分钟）。 </li></ol></li><li> 执行使用任何能够分离和检测的碳水化合物的HPLC系统TRET反应混合物的HPLC分析。该协议涉及到使用氨丙基HPLC柱和检测用折射率碳水化合物的分离。 <ol><li>附加包含柱前后卫的HPLC色谱柱氨丙基（4.6×250毫米）。 </li><li>以0.4的流量平衡以在去离子水中80％乙腈氨基丙基柱毫升/分钟。 </li><li>加载的反应产物（或标准）的到氨基丙基柱的溶液。 </li><li>以0.4毫升/分钟的流速和50℃的柱温洗脱产物（或标准），在去离子水中的80％乙腈。典型地，所使用的运行时间为40分钟。 注意:两个葡萄糖类似物原料和海藻糖类似物产物可通过折射率被检测到，尽管可以使用其他方法，如蒸发光散射检测器（ELSD）。使用所描述的条件下，葡萄糖类似物通常洗脱10-15之间15-25分钟之间分钟和海藻糖类似物洗脱。 </li></ol></li><li> 纯化海藻糖类似物的NMR分析。 <ol><li>溶解在D 2 O中（700微升）纯化海藻糖类似物，将溶液转移到NMR管中。 </li><li>获得1 H和根据适当NMR设施协议。13 C NMR光谱。 </li></ol></li></ol><ol><li> 合成，纯化和管理6 TreAz以耻垢分枝杆菌 （Msmeg）。 <ol><li>添加6-叠氮基-6-脱氧吡喃葡萄糖（6-GlcAz，0.020毫摩尔，4.1毫克），UDP-葡萄糖（0.040毫摩尔，24.4毫克）， 氯化镁 （0.020毫摩尔，4.1毫克）于15毫升锥形管中。 </li><li>添加TRET在Tris缓冲液（来自步骤1.5.4获得），以实现300微克/毫升的最终酶浓度和1毫升的最终体积。吸管将混合物上下轻轻或反转管以溶解固体。 </li><li>孵育在70℃振荡15分钟进行反应。 </li><li>稀释酶反应混合物用3毫升的去离子水，并将其转移到预先洗涤的离心过滤单元（NMWL 10kDa的）。在3000 xg离心离心过滤装置直到大部分液体通过过滤器进入管，大约10分钟。 </li><li>丢弃日离心过滤单元电子上议院。混合床离子交换树脂（0.75）加入到管中，搅拌/摇在室温下添加25分钟。倒出上清液并将其过滤以除去树脂。 注意:步骤5.1.1-5.1.5在不到1小时提供6-叠氮基海藻糖（6- TreAz）中的大约5毫浓度的水溶液。在5mM的浓度是基于底物向产物的定量转化，并且在纯化步骤需要的地方，在这些步骤中假设产物的损失最小稀释估计。该溶液可以是无菌过滤如果需要除了生物样品之前。 </li><li> 6- TreAz产物溶液适当体积添加到耻垢分枝杆菌的对数相培养（Msmeg），通常以达到100-1,000微升的最终培养物体积和25微米的〜最终-6- TreAz浓度。孵育细胞在37℃，所需的时间量，通常为60分钟。 </li></ol></li><li> 执行点击化学缀合荧光基团叠氮化标记的细胞。在这个协议中，使用铜-催化的叠氮化物-炔环加成（CuAAC），以在Msmeg递送荧光团与细胞表面叠氮化物。 <ol><li>离心细胞在3900×g离心5分钟，然后洗涤细胞用含有0.5％牛血清白蛋白的PBS。重复两次。 </li><li>重悬在4％的多聚甲醛的沉淀的细胞在PBS中解决这些问题。 10分钟温育后，重复步骤5.2.1至洗涤细胞。 </li><li>重悬在138微升PBS沉淀的细胞。 </li><li>添加在DMSO炔羧基罗丹明110（炔-488）的1mM储备液3微升。 </li><li>添加抗坏血酸钠在去离子水中新鲜制备的60毫原液的3微升。 </li><li>添加三[（1-苄基-1H-1,2,3-三唑-4-基）甲基]胺（TBTA）的叔丁醇/二甲亚砜6.4毫米原液（DMSO）中的3微升4:1。 </li><li>加入3＆＃956:L在去离子水中的CuSO 4的50mM储备液。 </li><li>吸管将细胞悬液上下，然后在黑暗中室温下培养30分钟。 </li><li>重复步骤5.2.1至洗涤细胞。重新悬浮在150μL的PBS中的细胞。 </li></ol></li><li> 进行细胞荧光分析。在这个协议中，使用荧光显微镜可视化标记Msmeg的细胞荧光。 <ol><li>加悬浮在PBS中至显微镜载玻片细菌细胞的10微升和液体轻轻扩散成使用盖玻片边缘的薄层。放置空气中的暗干燥。 </li><li>添加安装介质的10μL在干燥的样品，然后将盖玻片在样品和应用胶粘剂（ 如指甲油），以固定。 </li><li>图象使用放大100倍的荧光显微镜的幻灯片。 </li></ol></li></ol>

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The authors have nothing to disclose.

海藻糖类似物有冲击各个领域，从食物和药品的保存到微生物感染6的诊断和治疗的潜力。现有的多步化学合成方法是要修改后的多个站点生产复杂海藻糖类似物有用的（ 如 ，自然发生的复杂的分枝杆菌糖脂）。然而，这些方法不可避免地冗长和低效的，即使当施加到比较简单的单取代海藻糖类似物9，13，14的合成。需要替代的合成方法，以快速和有效地生产各种型号可以具有在上述领域值海藻糖类似物。即利用从自然海藻糖合成酶的生物催化方法保存最有希望。尽管一些海藻糖合成途径存在于自然界中，我们考虑TRET从T. TENAX 18成为海藻糖类似物合成提供了理想的酶有几个原因。 TRET从T. TENAX是热稳定的，允许反应进行的速度提高，在大规模生产设置预防微生物污染的进行加热。 TRET从T. TENAX是单向的酶是不能够降解海藻糖。从TRET TENAX T.是适合于大肠杆菌表达和纯化，其运输和储存是出类拔萃的。该TRET反应是一个单一的步骤，它涉及简单的底物葡萄糖和UDP葡萄糖。大量的结构/功能多样葡萄糖类似物可以从众多的商业供应商。 UDP-葡萄糖是一种相对便宜的葡萄糖供体，特别是相比于TREP施主β-D-葡萄糖1-磷酸。从TRET TENAX T.具有较高的PRomiscuity为葡萄糖衬底结构​​，所以各种海藻糖类似物是可访问的。从TRET反应混合物产物纯化是快速和简单。 在此，我们报道了海藻糖类似物合成的详细优化的协议，关于TRET酶的上述积极属性大写。该TRET方法可用于访问在半制备规模（10-100毫克）的各种纯海藻糖类似物，虽然如果需要附加的放大是可能的。多种类型的类似物在我们之前的和目前的工作，包括叠氮基，脱氧，氟- ，和硫代海藻糖，以及海藻糖立体异构体访问，但是其他的修改也将是可能的（ 例如 ，稳定同位素标记和放射性标记的海藻糖是特别感兴趣的）。该TRET过程的合成和纯化步骤是在操作简单，可以很容易地通过谁不合成澈经过培训的人员进行米斯特里。之一的TRET过程的最吸引人的特点是，无论是合成和纯化步骤可以在一段很短的时间取决于所需的（注的洗涤步骤的数目来执行，从1-4小时:如果尺寸排阻柱需要纯化到海藻糖类似物产物从未反应的葡萄糖类似物分开，这增加了总时间约2天，这仍然是基本上大于海藻糖类似物合成现有方法）更快。 虽然TRET过程是访问尚未报告海藻糖类似物的最有效途径酶，它有一些缺点，这为未来的改进机会。首先，尽管相对于其他糖供体的UDP葡萄糖是相对便宜的，所述TRET过程的效率可以进一步通过用来自便宜的来源UDP-葡萄糖的酶的合成， 例如 ，α-D-葡萄糖-1-耦合它改善磷酸盐，或由identifyi纳克UDP葡萄糖一种廉价的替代品。这种方法的第二个潜在的局限性是反应范围最终由TRET的衬底公差约束。而野生型TRET的公差是相当高的（ 见表1），某些类似物不能使用该方法（ 例如 ，4位上修饰的类似物）合成。因此，增加基底宽容TRET的设计版本将是有价值的。第三，虽然许多TRET催化反应以定量转化继续，较低的转化就必须一个色谱纯化步骤。在这个协议中，我们将重点放在使用相对慢的大小排阻过程，因为它价格低廉，不需要任何专门的设备，并且可以用水洗脱单独进行。可替代地，采用制备规模氨基丙基柱HPLC纯化，可以用作用于产物纯化更快方法。尽管其中的一些限制，在TRET过程提供一个强大的平台海藻糖类似物的快速，高效的准备，而且应该加快其在生物（TECHNO）评估和应用逻辑和生物医学领域。未来改进的TRET过程中，上面所讨论的，将进一步加强研究和相关的海藻糖及其衍生物的应用程序。 海藻糖类似物在各个领域的潜在价值。如在引言中提到的，天然海藻糖用于biopreservation和食品甜味剂的应用，所以海藻糖与诸如降解抵抗性类似物可以是有吸引力的。另外，各种天然海藻糖稳定同位素标记和放射性标记的版本是可商购用于研究目的，并且TRET过程当然可以提供给这些分子快速和高效的访问。海藻糖类似物是以来海藻糖哺乳动物针对缺席与探头和抑制剂病原体的兴趣也。一个AP折襞在这方面是特别令人感兴趣的是结核分枝杆菌的检测。 结核分枝杆菌 ，结核的病原体，其每年杀死150万人，包含对哺乳动物缺席独特海藻糖处理途径。例如，海藻糖回收运送器和结合了海藻糖混入外膜驻留糖脂下游酶不存在于哺乳动物22。针对这个特定的分支杆菌，机械用检测的海藻糖类似物表示在模型系统中，可能的人类患者有吸引力的方法，以在体内 的结核分枝杆菌感染的成像。事实上，FITC酮海藻糖和TreAz（ 图1B）的上述实施例已经铺平了道路这类发展8,9。为了方便这些工具来研究团体访问，我们在此报道迅速协议树脂工艺条件的ING和采用6 TreAz图像分枝杆菌结合点击化学。 这个协议可以适于其它可检测的海藻糖类似物，可能是结核病的诊断应用中有用的发展。例如，Barry和戴维斯首次提出了可能使用18 F-氟-海藻糖作为正电子发射断层扫描（PET）探测器对分枝杆菌感染8的体内成像。该TRET过程可能是理想的用于实现这一目标。 TRET能够由2-氟-葡萄糖快速定量生成2-氟-海藻糖（ 见表1），这是至关重要的，因为（18）F-2-氟-葡萄糖是短命（18 F-半衰期= 110分钟），因此需要极其快速的化学反应。此外，由于它是在临床上用于肿瘤的PET成像已经使用18 F-2-氟-葡萄糖是现成的。使用所描述的TRET过程的dev的18架F-氟海藻糖elopment和各种应用等海藻糖类似物目前在我们的实验室进行。

海藻糖是由由1,1-α，α-糖苷键（ 图1A）连接的两个葡萄糖部分的对称非还原二糖。而海藻糖是从人类和其他哺乳动物不存在，它是在细菌，真菌，植物和无脊椎动物1常见。海藻糖在大多数生物体的主要作用是为了防止环境压力，如干燥1。另外，一些人病原体需要海藻糖毒力，包括结核引起结核分枝杆菌 ，它利用海藻糖作为细胞膜的生物合成的介体和作为一个积木为免疫调节糖脂2的结构。 <img alt="图1" src="/files/ftp_upload/54485/54485fig1.jpg" /> 图1: 海藻糖和海藻糖类似物。 （A天然海藻糖和非天然海藻糖类似物，其中X是一个结构修改的）结构。在具有如biopreservation和生物成像的潜在应用的文献报道海藻糖类似物（B）的实施例。 由于其独特的结构和生理功能，海藻糖已经引起显著悉心生物（TECHNO）使用逻辑和生物医学应用3。在以自然观察如海藻糖的保护性能，其惊人的能力，以帮助维持生命在经历了极度脱水4 -具备刺激了biopreservation应用中的广泛使用的"复活"的植物。海藻糖已被用于保持一个宽的生物样品，如核酸，蛋白质，细胞和组织3个这样的阵列。例如，海藻糖在许多药物t一起使用作为稳定添加剂帽是在市场上，包括几个抗癌单克隆抗体3。还有，海藻糖用作在食品工业中的甜味剂，它广泛地用于在两个食品和化妆品工业产品保存。海藻糖为这些类型的商业应用的采用最初是由无法从天然来源或通过合成获得纯海藻糖的大批量的限制。然而，对于经济的生产从淀粉海藻糖的有效酶促过程最近被开发出来，这刺激了其广泛的商业用途 5。 化学修饰的海藻糖的衍生物，在此称为海藻糖类似物，都获得了越来越多的关注的各种应用（在图1A中所示的通用结构; 图1B示出的海藻糖的类似物的具体例子）6。例如，乳海藻糖海藻糖类似物与半乳糖代替其葡萄糖单位之一，因此它的4-位羟基具有倒置立体化学构。乳海藻糖具有相同的稳定性质海藻糖而是降解抗性通过肠酶，使得它作为一种无热量的食品添加剂6,7吸引力。 本集团的海藻糖类似物的兴趣，主要涉及他们的具体分枝杆菌探针和抑制剂的价值。巴里和Davis组开发了荧光素共轭酮海藻糖类似物，命名为FITC酮基海藻糖，这被证明代谢标记的活结核分枝杆菌的细胞壁，通过荧光显微镜8启用其检测。所述Bertozzi教授实验室开发小叠氮基海藻糖（TreAz）类似物，可以代谢标记细胞壁，随后是DETected使用点击化学和荧光分析9。这些进步指向使用基于海藻糖探针作为结核病诊断显像剂的可能性。海藻糖类似物也已实行如结核分枝杆菌的抑制剂由于其潜在的破坏在用于活性和毒力10，11，12所必需的细菌通路。 到目前为止，主要障碍显影海藻糖类似物的生物（TECHNO）逻辑和生物医学应用是缺乏有效的合成方法。这两个传统路线，以生产海藻糖类似物依靠化学合成（ 图2）。一种途径涉及天然海藻糖desymmetrization /修改，而​​另一个涉及开始适当官能化单糖积木并且执行化学糖基化，以伪造1,1-α，α-糖苷键。这些方法中，最近已在综述文章13,14所讨论的，已经证明了从结核分枝杆菌 15完成小批量的复杂含海藻糖的天然产品，例如sulfolipid-1的多步合成是有用的。然而，这两种方法一般是低效率的，耗费时间，不能访问非化学家和，另外，不被认为是环境友好的。因此，用于合成某些类型的海藻糖类似物，这些策略是不理想的。 <img alt="图2" src="/files/ftp_upload/54485/54485fig2.jpg" /> 图2: 途径海藻糖合成类似物。化学方法对海藻糖合成类似物，如左图所示，使用包括难以PROTEC多步程序化/去保护，desymmetrization，和/或糖基化的步骤。酶法合成，如右图所示，使用酶，以立体转换简单，未受保护的基材海藻糖在水溶液中的类似物。本文所报道酶促协议使用海藻糖合酶（TRET）酶对葡萄糖类似物和UDP-葡萄糖转化成海藻糖类似物在一个单一的步骤。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 海藻糖类似物的高效生物催化路线将促进生产，评价，这个充满希望的类分子的应用。而海藻糖生产5商用酶促过程并不适用于合成的类似物，因为它利用淀粉作为底物，还有其它生物合成路径在大自然的方式，可为海藻糖合成类似物被利用。但是，这方面的研究，这是最近点评 ，是有限的。一份报告使用的大肠杆菌海藻糖合成途径的启发方法，从相应的氟葡萄糖访问单个氟海藻糖类似物。然而，这种方法需要一个三酶系统具有有限的效率和通用性8。已探索了另一种方法是在相反方向，这在原理允许从葡萄糖类似物和葡萄糖-1-磷酸6，16，17海藻糖类似物的一步合成使用海藻糖磷酸酶（TREP）。虽然这种方法可能有未来的承诺，反相以及留住TrePs目前有用于模拟合成的缺点。例如，反相TrePs具有令人望而却步EXPEnsive供体分子（β-D-葡萄糖-1-磷酸）和保留TrePs具有差的酶表达产率/稳定性和有限的基板滥交。显著的改善（ 例如 ，通过酶工程）将前TREP介导的模拟合成需要的是实用的。 目前，对于海藻糖类似物的酶促合成的最实际的方法是使用一个海藻糖合酶（TRET）酶，它转换葡萄糖和尿苷二磷酸（UDP）的葡萄糖转化为海藻糖在一个单一的步骤6。我们最近报道了使用Thermoproteus TENAX TRET-热稳定和单向酶18的-to从葡萄糖类似物和UDP-葡萄糖（ 图3）19合成海藻糖类似物。这种酶只操作在合成方向，并且避免在TREP系统中发现海藻糖降解的问题。这一步反应库仑ð在1小时内完成，并且各种各样海藻糖类似物在高产率被访问从容易获得的葡萄糖类似物基板（ 见表1中的代表性的成果（高达&gt; 99％为通过高效液相色谱法（HPLC）测定）部分）。 <img alt="图3" src="/files/ftp_upload/54485/54485fig3.jpg" /> 图3: 海藻糖类似物TRET催化一步法合成。从T. TENAX所述TRET酶可立体选择性加入现成的葡萄糖类似物和UDP-葡萄糖以形成海藻糖类似物中的一个步骤。 R 1〜R 4 =可变结构修饰，例如叠氮，氟，脱氧，硫代，立体，或同位素标记的修改; Y =可变的杂原子，例如氧或硫，或同位素标记的杂原子。 在这里，我们提供的广告etailed协议用于TRET合成过程中，其中包括从大肠杆菌表达和TRET的纯化，优化TRET反应条件，那就是在水相中完全进行的改进纯化方法。该修改协议使得在半制备规模（10-100毫克）多样海藻糖类似物的权宜和高效合成和纯化。我们还证明了制备和在不到1小时，这使分枝杆菌细胞的快速荧光检测施用基于海藻糖探针分枝杆菌使用该协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LB agar</td>
			<td>Research Products International</td>
			<td>L24021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria broth</td>
			<td>Research Products International</td>
			<td>L24045</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth</td>
			<td>Research Products International</td>
			<td>T15050</td>
			<td></td>
		</tr>
		<tr>
			<td>L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A3256</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered Saline</td>
			<td>GE Healthcare</td>
			<td>SH30256</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BDH</td>
			<td>BDH9286</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate,</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">S374</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>monobasic</td>
		</tr>
		<tr>
			<td>Syringe filter, 0.45 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>09719D</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor mini-tablets, EDTA-free</td>
			<td>Thermo Scientific</td>
			<td>88666</td>
			<td></td>
		</tr>
		<tr>
			<td>HisTrap HP nickel affinity column, 5 mL</td>
			<td>GE Healthcare</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS base ultrapure</td>
			<td>Research Products International</td>
			<td>T60040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing, MWCO 12&#8211;14,000</td>
			<td>Fisher Scientific</td>
			<td>21-152-16</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Glucose analogues</td>
			<td>CarboSynth,</td>
			<td rowspan="3"></td>
			<td rowspan="3">Examples of vendors that offer numerous glucose analogues</td>
		</tr>
		<tr>
			<td>Sigma Aldrich,</td>
		</tr>
		<tr>
			<td>Santa Cruz Biotechnology, American Radiolabeled Chemicals</td>
		</tr>
		<tr>
			<td>6-Azido-6-deoxy glucopyranose (6-GlcAz)</td>
			<td>CarboSynth</td>
			<td>MA02620</td>
			<td></td>
		</tr>
		<tr>
			<td>UDP-Glucose</td>
			<td>abcam Biochemicals</td>
			<td>ab120384</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate&#160;</td>
			<td>Fisher Scientific</td>
			<td>M33</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 centrifugal filter unit</td>
			<td>EMD Millipore</td>
			<td>UFC901008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rex RG 501-X8 mixed-bed ion-exchange resin</td>
			<td>Bio-Rad</td>
			<td>444-9999</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra-Fine Bio-Gel P2 media</td>
			<td>Bio-Rad</td>
			<td>150-4118</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-backed silica gel thin-layer chromatography plates</td>
			<td>EMD Millipore</td>
			<td>1056280001</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Butanol</td>
			<td>Fisher Scientific</td>
			<td>A399</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>S25310A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fisher Scientific</td>
			<td>A300</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>EMD Millipore</td>
			<td>AX0145</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide, 99.8%</td>
			<td>Acros Organics</td>
			<td>351430075</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminopropyl HPLC column</td>
			<td>Sigma Aldrich</td>
			<td>58338</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Para-formaldehyde</td>
			<td>Ted Pella</td>
			<td>18505</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkyne-488</td>
			<td>Sigma Aldrich</td>
			<td>761621</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td>Click Chemistry Tools</td>
			<td>1061</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butanol</td>
			<td>Sigma Aldrich</td>
			<td>360538</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>W387520</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate</td>
			<td>Sigma Aldrich</td>
			<td>C1297</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting medium</td>
			<td>Southern Biotechnology</td>
			<td>10001</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid one-step enzymatic synthesis and all-aqueous purification of trehalose analogues

化学修饰的海藻糖，或海藻糖类似物的版本，在生物学，生物技术和制药科学应用，其他领域中。例如，海藻糖类似物轴承可检测标记已被用于检测结核分枝杆菌 ，并且可以具有应用程序作为结核病诊断显像剂。海藻糖水解稳定版本也正在采取由于其用作无热量甜味剂和生物保护剂的潜力。尽管这类的各种应用的化合物的吸引力，其潜在的由于缺乏对他们的生产一个健壮的路由的仍未实现。在这里，我们报告海藻糖类似物的快速，高效的生物催化一步合成绕过与化学合成相关的问题进行详细的协议。通过利用从Thermoproteus TENAX耐高温海藻糖合酶（TRET）酶，海藻糖类似物可以是发电机密封编在从葡萄糖类似物和高收率尿苷二磷酸葡萄糖单一步骤在15-60分钟（不超过定量转化）。一个简单的和快速的非色谱纯化协议，它由旋透析和离子交换的，可以在水溶液中在短短的45分钟递送已知浓度的许多海藻糖类似物。在未反应的葡萄糖类似物仍保持的情况下，可以进行海藻糖类似物产物的色谱纯化。总体而言，该方法提供了快速合成和海藻糖类似物纯化即是有效的，可访问的非化学家"绿色"生物催化的平台。为了举例说明了该方法的适用性，我们描述了一种协议，用于合成，所有水的净化，和基于海藻糖点击化学探针分枝杆菌，所有这些都用了不到1小时，使分枝杆菌的荧光检测的施用。在未来，我们设想，除OTH呃应用中，该协议可以被应用到基于海藻糖探针结核病诊断的快速合成。例如，短寿命核素改性的海藻糖类似物（ 例如 ，（18）F-改性海藻糖）可用于先进的临床成像方式如正电子发射断层扫描，计算机断层扫描（PET-CT）。

T. TENAX TRET从大肠杆菌中的大约4毫克使用标准蛋白质表达和纯化技术/ L的产率获得。单个镍亲和层析步骤足以从大肠杆菌裂解物纯化TRET（代表性FPLC跟踪示于图4）。如建立了我们对TRET合成过程初始公布的，重组的T。TENAX TRET能够转换各种各样葡萄糖类似物-其中许多的是可商购到相应的海藻糖类似物以高效率19。 表1，它给出的HPLC确定反应产率的数目开始使用我们的最初报告协议葡萄糖类似物，示TRET的衬底滥交及其合成的适宜的范围。不同的结构修改，包括氟，脱氧，叠氮，硫基，并且在糖环周围的不同位置的立体化学修饰是由野生型酶耐受性良好，与主要的例外是在4-位上的修改。 <img alt="图4" src="/files/ftp_upload/54485/54485fig4.jpg" /> 图 4: 通过FPLC TRET纯化代表性的结果。从大肠杆菌裂解液的重组T. TENAX TRET的纯化进行了使用如在步骤中描述1.4.1-1.4.4镍亲和层析。蓝，紫外（UV）吸光度跟踪;浅绿色，洗脱缓冲液的浓度;深绿色，压力。对应于TRET峰值是用箭头表示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 表1: 代表性的产量为TRET反应。 HPLC确定并连续海藻糖类似物TRET反应的分离产率。 ND，未检测到。 - 表示未使用优化的协议在半制备规模进行该反应。 *表示被要求的尺寸排阻层析步骤。横分割线分离修饰的海藻糖糖环上的位置。表改编自经许可基准19;更新，包括这项工作的分离产率。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/Table1.docx">请点击这里下载此文件。</a> 这里，我们提出优化，以我们的初始协议提高TRET合成过程的总效率和速度。而原来的协议使用10毫米的葡萄糖的alogue和40mM的UDP-葡萄糖，它被确定可比转化可以使用的20mM葡萄糖和40mM的UDP-葡萄糖来获得，从而有效地增加了一倍每个反应中产生的产物的量，并限制UDP-葡萄糖的浪费，这是相对昂贵的。使用等摩尔量的葡萄糖类似物和UDP葡萄糖导致较低的转换。如果需要的话，反应时间也可缩短至小于最初报道60分钟，同时仍保留可比变换为许多类似物，其通过6- GlcAz至6-TreAz的定量转化在仅15分钟，在70℃下表现出（ 图5和6）。 本文所报道的纯化方案大大改进，用非色谱自旋透析/离子交换法代替原酶沉淀/硅胶色谱法。对于本变形例的基本原理是，TRETř反应的影响完全混合组成，除了中性海藻糖模拟产品的离子种类。因此，该混合物可以是旋透析以除去酶，然后用混合床离子交换树脂处理以除去所有的离子种，在短短的45分钟递送纯化海藻糖类似物在水溶液中。此全含水纯化方法避免了对环境有害的有机溶剂，费时蒸发和过滤步骤，和干负载硅胶色谱，这是一个缓慢而繁琐的过程，特别是用于非化学家。对作为使用所描述的TLC或HPLC分析测定显示出定量转化TRET反应，该纯化方法提供了约60-80％的分离产率在所报告的反应规模，与产品的损失可能是由于在离子交换的一些糖结合树脂（参见代表分离产率见表1）。在未反应的葡萄糖类似物仍的情况下，它可以从用聚丙烯酰胺基于珠子的尺寸排阻柱所需海藻糖类似物产物进行分离，用水洗脱。如果优选的，这也可以使用制备规模的HPLC用氨基丙基柱来完成的。产品的纯度可通过薄层色谱法，HPLC和/或NMR光谱分析进行评估。 图5示出了用于6-TreAz，其经由所述协议合成代表性的分析数据。的TLC和HPLC数据表明6- GlcAz衬底定量转化至6- TreAz产品用于此反应，和自旋透析/离子交换纯化步骤后分离的产率是58％。 1 H和13 C-NMR光谱分析证实6- TreAz产品的结构，最值得注意的是，包括新形成的糖苷键的α，α-立体化学，以及它的纯度。 <img alt="图5" src="/files/ftp_upload/54485/54485fig5.jpg"/&gt; 图5: 为TRET反应代表的分析数据。 （A）的薄层和（B）6- GlcAz至6-TreAz TRET催化转化的HPLC分析。 （C）的1 H NMR和（D）。13 C NMR自旋透析/离子交换纯化后获得的6- TreAz产物的光谱。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 定海藻糖类似物的结核分枝杆菌的特异性检测上述值，则TRET过程应方便结核病研究和诊断应用的开发和使用基于海藻糖探针。为了证明这种类型的应用中，我们使用的优化TRET过程迅速制备，纯化和管理6- TreAz-基于海藻糖点击化学探针9 -to分枝杆菌以使荧光检测（工作流和有代表性的数据被在图6中示出）。简言之，TRET用于商业-6- GlcAz转化为6- TreAz定量在15分钟，然后将反应混合物进行纺丝透析/离子交换纯化方法，该方法只用了45分钟（省略多个漂洗步骤，以增加速度）。因此，在仅1小时内产生的已知浓度的纯6- TreAz（〜5毫米）的水溶液原液。 6- TreAz库存立即施用到模型细菌Msmeg（最终6- TreAz浓度〜25微米）的生长培养物来完成细胞表面，其中嵌入一个手柄的点击化学介导的结扎20，21的叠氮化物标记荧光探针。后来，标记的细胞通过荧光显微镜，其显示强荧光为6- TreAz处理的细胞进行分析。正如所料，未用6- TreAz或处理的对照样品缺少官能海藻糖传送器22，这是需要的6- TreAz摄取和代谢掺入9，显示没有荧光。 <img alt="图6" src="/files/ftp_upload/54485/54485fig6.jpg" /> 图6: 用于快速检测用TRET合成模拟海藻糖分枝杆菌的代表性成果。 （ 一 ）工作流的快速合成，纯化，并使用6滓的AZ获取Msmeg的荧光检测。步骤的协议的5.1.1-5.1.6用于合成和纯化6- TreAz（给予〜5mM的在1个小时的水溶液），然后施用它住Msmeg完成与叠氮化物在细胞表面的代谢标记。接着，在步骤5.2.1-5.2.9所述，点击化学（CuAAC）进行与炔改性的荧光团，炔-488反应的细胞表面叠氮化物。 6 TreAz处理Msmeg包含的功能海藻糖转运体（野生型和ΔsugC :: sugC）表现出较强的荧光，而Msmeg的对照样品不及时治疗或缺乏海藻糖转运（ΔsugC）没有（B）荧光成像。图改编自经许可基准19;从提高TRET协议的工作流程和成像数据进行更新。比例尺，5μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54485/54485fig6large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues at JoVE.com

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

快速一步酶法合成海藻糖类似物的全净化水

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

rapid-one-step-enzymatic-synthesis-all-aqueous-purification-trehalose

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.3K 次观看.  Central Michigan University. Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

视频: Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

科研

生物化学

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12的表达，纯化和抗菌活性

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

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

哺乳动物 Bestrophin 离子通道的表达与纯化

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 细胞 Mitoribosome 的快速分离

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

一种可定制的酶化生产与纯化天然产物的方法

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick，将膜蛋白一步进生物仿入肽，用于简化蛋白质结合测定

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...