作者要感谢路易斯·德尔加迪略先生（俄亥俄大学化学和生物分子工程系）、马修·威廉斯先生（俄亥俄大学化学和生物分子工程系）和菲利伯托·塞德诺-劳伦特博士（布里格姆妇女医院和哈佛医学院）提供专家技术援助。作者还要感谢2011年冬季CHE 404/BME 504和2012年秋季CHE 4830/BME 5830学生（俄亥俄州大学化学和生物分子工程和生物医学工程系）的技术咨询和讨论。这项工作得到了国家科学基金会（NSF）主要研究仪器赠款CBET-1039869（MMB）、NSF CBET-1106118（MMB）、皮肤病基金会研究补助金A050422（SRB）、国家卫生研究院（NIH）NCI赠款1R15CA161的支持 830-01 （MMB）， NIH 基尔希斯坦-NRSA 博士后奖学金 F32CA14219-01A1 （SRB）， NIH/NCI R01CA173610 （CJD） 和 NIH NCCAM 授予 R01AT004628 （CJD）。

1. 验证蛋白质 A 的亲和力
<ol><li>验证 Fc 表达蛋白（重组融合蛋白或 IgG 抗体， 以下简称"蛋白质"）在使用蛋白A膜吸入器之前，对蛋白质A14-17 具有可接受的亲和力，并测试蛋白质在库存溶液中的存在（例如 ，通过离心在1，000 x g中澄清的细胞培养超纳特，5分钟到颗粒悬浮细胞）。使用流细胞测量、西印、ELISA 等。根据实验室偏好检测 Fc 的蛋白质功能和/或存在。理想情况下，量化细胞培养超自然（第6步）中的蛋白质，以避免超过蛋白质A膜增聚能力。如果检测到蛋白质，则继续。</li>
</ol>2. 准备蛋白质膜增发剂和细胞培养超自然
<ol><li>按照制造商的说明准备膜吸食器。 切勿让空气进入膜沉着器。通过膜吸食器灌注的所有溶液应在室温下使用 0.22 μm 过滤器进行预过滤。 用 0.22μm 过滤的杜尔贝科磷酸盐缓冲盐水 （DPBS） 或选择的缓冲液填充 10 毫升注射器，并释放气泡。香水 DPBS 以去除存储解决方案，并在使用前立即平衡膜增压器。</li>
	<li>使用真空驱动的 0.22μm 无菌过滤装置，在灌注前立即通过膜吸附器过滤细胞培养超自然物。</li>
</ol>3. 加载蛋白质膜增发器
<ol><li>将细胞培养超自然物吸进 Luer Lock 注射器（30 毫升或更大），并释放任何气泡。</li>
	<li>组装材料和设备，如 图1所示。将注射器连接到膜吸食器的入口。将柔性管连接到膜吸食器插座上。如有必要，在注射器和膜吸食器之间放置一个 0.22μm 注射器过滤器。使用烧瓶或瓶子从吸食者处捕获流经（也称为过滤）。</li>
	<li>将注射器泵设置为所需的体积流量，但不超过制造商推荐的流量（例如 10 毫升/分钟）。通过膜增压器将细胞培养超自然体灌注，收集通过烧杯或其他容器的流量。将注射器重新装上所需的超标剂。</li>
	<li>如果需要，使用流动细胞测量、西印、ELISA 等测试蛋白质的流动。基于实验室偏好。通常没有必要反驳流过的流程。</li>
</ol>4. 膜除胶器中的埃卢特蛋白
<ol><li>用 10 毫升 DPBS 清洗膜吸食器，以去除任何非绑定蛋白。</li>
	<li>使用 10-15 毫升的洗涤缓冲器（例如基于胺的洗涤缓冲器（pH 2.8）时，以所需的流速（例如1 毫升/分钟）从膜吸食器中提取的 Elute 蛋白质。在含有中和缓冲器（例如1 M Tris （pH 9.4） 的管中捕获提升，其容量为 10%（1.0-1.5 毫升）。</li>
	<li>或者，将一毫升增量中的蛋白质放入含有 100 μl 中和缓冲液的管中，使用总容量为 10-15 毫升的洗脱缓冲器。使用首选的表征方法测试每个分数中蛋白质的存在。</li>
	<li>根据制造商的说明再生膜沉着器。香水 10 毫升 0.22 μm 过滤 DPBS 或选择缓冲区，然后 10 毫升 0.22 μm 过滤 50 m NaOH 在 1 N NaCl，最后 10 毫升 0.22 μm 过滤 DPBS。在 DPBS 中填充膜吸食器，在 DPBS 中填充 20% 乙醇，以在 4 oC 下进行长期存储。</li>
</ol>5. 浓缩和透析蛋白质
<ol><li>将所有高放（或在第 4.3 步确定的含有蛋白质的浓缩分数）沉积在 10 kDa 分子重量截断离心滤清器单元中。按照制造商的离心指示操作。</li>
	<li>按照制造商的说明，在小容量透析单元（10 kDa 分子重量切断）中对选择的缓冲区进行透析。如果蛋白质沉淀是一个问题，则可能需要在透析材料中添加缓冲区。如果需要，透析材料可以冷藏，直到第 6 步可以执行，但不建议长期存储，不含防腐剂。</li>
</ol>6. 在快速的西方膨胀程序中量化和描述纯化产品
<ol><li>在18-20的减少或不降低条件下，通过 SDS-PAGE 在选择的凝胶上解决纯化蛋白和 Fc 标准。使用迷你凝胶而不是米迪凝胶，以尽量减少运行时间。<ol>
<li>如果尚不为人知，则确定 Fc 标准范围，在加载量（例如 0.1-1 毫克）方面，波段信号的线性变化。使用相同的凝胶、运行条件、转移条件和试剂，用于量化和描述纯化蛋白质。</li>
			<li>加载多种纯化蛋白样本，包括用 DPBS 稀释的样品，以确保纯化蛋白带信号在图像分析中 Fc 标准的线性信号范围内。</li>
		</ol>
</li>
	<li>按照blotter制造商的指示，使用半膨胀的13将蛋白质从凝胶转移到不连续缓冲系统中的聚乙烯氟化物（PVDF）膜。传输时间通常为 5-10 分钟。如有需要，库马西在转移后弄脏凝胶，以验证转移效率21。</li>
	<li>使用真空辅助蛋白检测系统，根据实验室偏好，使用碱性磷酸酶 （AP） 或马萝卜过氧化酶 （HRP） 进行与抗 Fc 或抗 IgG 结合的免疫凝血。免疫印迹时间通常小于1小时。</li>
	<li>使用选择的基板和方法（如 AP基板和增强的化疗）开发免疫体。</li>
	<li>通过图像分析量化纯化蛋白。在来自 Fc 标准的波段信号上执行线性回归。如果 R2>0.90，则使用该线的方程计算其波段信号中的蛋白质数量。如有必要，考虑样品稀释。由于蛋白质的定量是在Fc的基础上进行的，调整蛋白质和Fc之间的分子重量差异值。 如果R2<0.90，则重复整个步骤6。</li>
	<li>使用功能检测或方法验证蛋白质，通过流动细胞测量、西斑、ELISA 等检测 Fc 。基于实验室偏好。</li>
	<li>将蛋白质稀释到所需的浓度和/或在纯化蛋白溶液中加入任何所需的防腐剂或稳定剂，然后再进行长期储存、冷冻或其他补充。</li>
</ol>

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没有什么可透露的。

此处描述的协议旨在快速分离和描述表达人类 Fc （Gal-1hFc） 的奇美融合蛋白，同时不会显著影响产品回收或膨胀成本。流线型工作流程的关键组件是用于隔离的蛋白质 A 膜吸附剂，以及用于西式印迹特征的半干转移和真空辅助免疫印迹。
Gal-1hFc 的隔离和特征处理时间从 300 毫升细胞培养超纳特（流线型协议中大约 9 小时，而传统协议中为 20 小时以上）的处理时间的急剧下降?...

使用蛋白质 A 亲和色谱法可以分离来自细胞培养超纳特和稀释裂解细胞溶液的抗体和重组蛋白表达免疫球蛋白 G （IgG） Fc 片段。在基础科学和工程实验室和工业领域，尽管成本高，加工时间长1-3，但富含多孔蛋白A涂层糖、玻璃或聚合物珠子的柱子最常用于亲和色谱。众所周知，亲和色谱是实验室和工业环境中最大的费用和处理瓶颈。因此，为了降低成本和加工时间，在不牺牲从工艺列车1，2，5-7的整体产品回收方面，已经进行了许多改进。抗体和其他Fc表达蛋白的分离特别有希望的是使用蛋白质A膜吸收剂2，5，7-10的亲和色谱。而柱色谱中的 Fc 捕获是扩散限制的（即Fc 表达蛋白必须扩散到内部毛孔并穿过内部毛孔才能到达大多数蛋白质 A），而高压在柱子上下降，膜吸收器中的大量传输由散装对流驱动，从而避免了扩散限制8，9，11，12。此外，膜增压器的压力下降较低，因此与珠子包装柱8，9，11，12相比，进液流速更快。因此，对于实验室规模隔离，膜色谱学预计将减少几个小时的隔离时间，并增加产品捕获量相比，列色谱。此外，还提出了膜吸附器中IgG吸附的数学模型8，9，11，12，从而使最终用户能够预测实验室的性能。
基础科学和工程实验室的另一个瓶颈是Fc表达蛋白的特征。然而，选择适当的方法来完成定性检测，如西方的污点，可以大大减少花费在产品测试上的时间。例如，在不连续的缓冲系统中进行半干旱转移可以在几分钟内完成蛋白质从 SDS-PAGE 凝胶到聚乙烯二氟化物 （PVDF） 膜的电泳传输，而不是在连续缓冲系统13中 1-2 小时的罐（湿）转移。
这里描述了一种快速、廉价和有效的协议，以分离和描述表达人类IgG的Fc片段的奇妙融合蛋白。大多数设备在标准基础生物医学科学、生物学、化学和（生物）化学工程实验室中都随时可用，所有试剂均市售。虽然具有代表性的结果来自粘液加洛汀-1/人类Fc幻想融合蛋白（Gal-1hFc）的分离和特征，但流线型协议可以应用于其他具有Fc片段的分子的分离，如抗体、Fc片段本身或其他Fc融合蛋白。与标准方法相比，我们的改进显著缩短了处理时间（只有 4 小时，但通常为 9 小时，而工作时间为 20 小时以上），同时实现了类似的或改进的产品恢复。

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Protein A membrane adsorber (Sartobind Protein A 2 ml)&#160;</td>
			<td style="width:191px;">Sartorius-Stedim</td>
			<td style="width:147px;">93PRAP06HB-12--A</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td>
			<td style="width:191px;">Millipore</td>
			<td>UFC801008</td>
			<td>Use 15 ml volume if needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sterile vacuum filter unit, 0.22 &#181;m, 500 ml</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">10 ml Syringes with Luer Lock tips</td>
			<td>BD</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">30 ml Syringes with&#160; Luer Lock tips</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tygon lab tubing (1/16 in x 1/8 in x 50 ft)</td>
			<td>Cole Parmer</td>
			<td>WU-95903-16</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td>
			<td>Thermo Scientific</td>
			<td>69576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. antibody collection tray</td>
			<td>EMD Millipore</td>
			<td>WBAVDABTR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. single well blot holder</td>
			<td>EMD Millipore</td>
			<td>WBAVDBH01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Elution buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tris, ultra pure</td>
			<td>Fisher Scientific</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">DPBS</td>
			<td>Thermo Scientific&#160;</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">DPBS with Ca2+/Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Human Fc</td>
			<td>Bethyl Research Laboratories</td>
			<td>&#160;P80-104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-APC</td>
			<td>Jackson Immunoresearch</td>
			<td>109136170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-AP</td>
			<td>Bio-Rad</td>
			<td>170-5018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Alkaline phosphatase substrate</td>
			<td>Promega</td>
			<td style="width:147px;">S3841</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Flow cytometry tubes (5 ml polystyrene or polypropylene)</td>
			<td>BD</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;"></td>
			<td style="width:191px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>55-2226</td>
			<td style="width:173px;">A peristaltic pump is also acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Protein gel electrophoresis system&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>552BR094876</td>
			<td style="width:173px;">Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Semidry blotter&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td style="width:147px;">690BR006163</td>
			<td style="width:173px;">Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:313px;">SNAP i.d. vacuum-assisted protein detection system</td>
			<td style="width:191px;">EMD Millipore</td>
			<td>WBAVDBASE</td>
			<td style="width:173px;">An upgraded model has replaced this specific instrument, but either should work just as well</td>
		</tr>
	</tbody>
</table>

isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow

实验室规模到工业规模净化生物分子从细胞培养超纳特和溶解细胞解决方案可以完成使用亲和力色谱。虽然使用多孔蛋白A的生长珠包装在列中的亲和力色谱可以说是实验室规模隔离抗体和重组蛋白表达IgG片段的最常见方法，但它可能是一个耗时且昂贵的过程。时间和财政拮据在小型基础科学实验室尤其令人生畏，这些实验室必须从稀释溶液中回收数百微克至毫克的蛋白质，但缺乏获得高压液体输送系统和/或具有生物分离专业知识的人员。此外，产品量化和定性也可能在多个工作日过长加工时间，并膨胀费用（消耗品、工资 等）。因此，对于实验室规模隔离和抗体和其他具有 Fc 片段的蛋白质的特征，需要快速、廉价但有效的协议。为此，我们设计了一个协议，由经验有限的技术人员在不到9小时（大约一个工作日）和最快4小时，而不是传统的方法，需要20多工作小时完成。大多数所需的设备在标准生物医学科学、生物化学和（生物）化学工程实验室中随时可用，所有试剂均市售。为了证明这一协议，提出了具有代表性的结果，其中用一种蛋白质A膜增聚剂分离出从细胞培养超母体中融合到人类Fc（Gal-1hFc）的奇美穆林甘胶-1。纯化Gal-1hFc采用西方快速印迹分析程序进行量化，并采用流动细胞学进行特征分析。流线型工作流可用于其他 Fc 表达蛋白（如抗体）和/或更改，以纳入替代定量和定性方法。

简化的Fc表达蛋白分离和特征协议通常用于处理从稀释细胞培养超纳特人融合到人类Fc（Gal-1hFc）的奇美粘膜粘膜加洛汀-1。 图 2 中的流程图说明了协议中每个步骤的工作流程和时间。对于典型的 300 毫升超纳特批次，执行弹性分数的可选流细胞学测试时，总处理时间约为 9 小时。如果省略了所有流细胞分析，由于缺少流量细胞仪或单独的实验室偏好，总处理时间可以短至 4 小时。一般...

Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

与传统的亲和色谱学相比，使用蛋白质A agarose珠状柱，蛋白质A膜吸收剂可以显著加快抗体和其他Fc片段表达蛋白的实验室规模隔离。适当的分析和量化方法可以进一步加快蛋白质加工，使隔离/定性可以在一个工作日内完成，而不是超过20个工作小时。

使用蛋白质膜吸收器和流线型工作流的奇美人 Fc 表达蛋白质的隔离和特征

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

isolation-characterization-chimeric-human-fc-expressing-proteins

Research

JoVE Journal

Bioengineering

7.1K 次观看.  Ohio University. 与传统的亲和色谱学相比，使用蛋白质A agarose珠状柱，蛋白质A膜吸收剂可以显著加快抗体和其他Fc片段表达蛋白的实验室规模隔离。适当的分析和量化方法可以进一步加快蛋白质加工，使隔离/定性可以在一个工作日内完成，而不是超过20个工作小时。

视频: Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

使用光声血流循环黑色素瘤细胞的检测和隔离

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

内含肽介导的人工蛋白水凝胶的合成

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

人体软组织的生物力学特性采用压痕和拉伸试验

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

含有宏观结构的光学组织幻影的制备与表征

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...