感谢国立台湾海洋大学海洋生物学研究所宏博士提供3种质粒结构。这项研究得到了科学和技术部 (大多数) 的赠款 106-2311-B-197-001 的支持。

1. 筹备工作
<ol><li>昆虫细胞培养<ol>
<li>准备50毫升细胞培养基。为此, 添加500µL 抗生素 (两性霉素 B = 0.25 µg/毫升, 青霉素 = 100 单位/毫升, 链霉素 = 100 µg/毫升) 和5毫升的热灭活胎牛血清在无血清细胞培养培养基 (不含血清或抗生素)。 注意:使用前, 在水浴中将胎牛血清加热65摄氏度, 30 分钟。</li>
		<li>维护斜纹 frugiperda (鳞翅目: 夜蛾科), sf9 昆虫细胞。通过摇动烧瓶并在光镜下检查, 从25厘米2细胞培养瓶中分离出约80% 的细胞。然后, 将细胞悬浮液的50% 转移到新的25厘米2细胞培养瓶中, 并允许细胞在室温下附着15分钟。用5毫升新鲜细胞培养培养基取代培养基, 在28摄氏度的孵化器中生长。通道细胞每2到3天取决于细胞的生长。</li>
	</ol>
</li>
	<li>细胞转染用质粒的制备<ol>
<li>将目标 DNA 片段 (例如, 毒蛾MNPV (LyxyMNPV) iap3基因及其删除构造) 插入 pDHsp/V5-His, 方法是基于 PCR 的克隆法11。利用 pcr 主配比 (2X) 和 pDhsp-F2/Op-IE2R 底漆集, 对转化后菌落的生长进行了菌落 pcr 检查。通过商业测序服务确认质粒序列。 注意:表 1列出了用于 PCR 的引物和相应的构造。</li>
		<li>培养单序列细菌菌落, 其中包含上述质粒结构 (步骤 1.2) 在200毫升 LB 培养基含有选定的抗生素 (50 µg/毫升), 分别。</li>
		<li>根据制造商的说明12, 从培养的大肠杆菌中提取质粒。</li>
	</ol>
</li>
	<li>用1.5 毫升细胞培养培养基和8.5 毫升无血清细胞培养培养基制备电镀培养基。</li>
	<li>放线菌 D (ActD) 细胞培养培养基, 加入1.5 µL ActD (1 毫克/毫升) 到10毫升细胞培养培养基 (最终浓度 = 150 ng/毫升)。存储在4摄氏度。</li>
</ol>2. 蛋白质瞬时表达
<ol><li>细胞播种<ol>
<li>通过摇动培养瓶来收割 sf9 细胞, 将细胞悬浮液倒进50毫升管, 并通过 P10 吸管将10µL 转移到 hemocytometer。在光显微镜下计数细胞数。</li>
		<li>板材 3 x 105 sf9 细胞入每个井在24井板材为15分钟在室温下。用0.5 毫升电镀介质替换介质。</li>
	</ol>
</li>
	<li>质粒的转染<ol>
<li>稀释细胞转染试剂: 稀释8µL 细胞转染试剂100µL 无血清细胞培养培养基, 并用涡流混合1秒。</li>
		<li>添加2µg 质粒 DNA (pDHsp70-Ac-P35/V5-His 或 pDHsp70-Ly-IAP3/V5-His 或 pDHsp70-Ly-IAP3-BIR/V5-His 或 pDHsp70-Ly-IAP3-RING/V5-His) 到100µL 的无血清细胞培养培养基和混合涡流 1 s (图 2)。</li>
		<li>将稀释质粒 DNA 与稀释细胞转染试剂 (210 µL) 相结合, 涡流 1 s. 室温孵育30分钟。</li>
		<li>添加210µL DNA 转染试剂混合物滴入细胞由 P1000 吸管。孵育28°c 为5小时。</li>
		<li>用 P1000 吸管取代电镀培养基, 用0.5 毫升的细胞培养培养基。用胶带封住24井板, 在28摄氏度孵化细胞16小时。</li>
	</ol>
</li>
	<li>热休克转染细胞: 把盘子放在42摄氏度的水浴中 (漂浮在水面上)。热30分钟, 并返回24井板28°c 孵化器。</li>
	<li>蛋白质表达的检测<ol>
<li>1小时或5小时热休克后, 用0.5 毫升 1x PBS 缓冲液冲洗细胞, 简要三次。 注: 将1毫升 10x pbs 缓冲器添加到9毫升灭菌 ddH2O, 将 10x pbs 缓冲器稀释为 1x pbs 缓冲器。</li>
		<li>溶解细胞与40µL 的 1x SDS 加载染料吹打上下。 注: 通过混合30µL 1x PBS 缓冲器和10µL 4x sds 样品缓冲器稀释 4x SDS 加载染料。</li>
		<li>在热块中将蛋白质样品加热98摄氏度, 10 分钟, 向下旋转1分钟, 然后放入冰上进行印迹测定。</li>
		<li>杂交印迹法 按照 Eslami 和 Lujan13中的西方印迹检测程序进行操作。运行 SDS 页凝胶14: 一凝胶受考马斯蓝染色 (加载控制, 以检查每个井中的蛋白质样品数量是否相等) 和其他受西方印迹检测, 根据 Eslami 和 Lujan13.</li>
		<li>检测 V5-tagged 融合蛋白与兔 anti-V5 抗体 (5 毫克/毫升) (1:5000 稀释 TBST 缓冲工作浓度1µg/毫升) 和山羊抗兔 IgG-辣根过氧化物酶 (HRP) 共轭 (0.8 毫克/毫升) (1:10000 稀释 TBST 缓冲工作浓度0.08 µg/毫升)。 注意:将聚丙烯酰胺的百分比调整为 17.5%, 当蛋白质分子量为 < 17 kDa 时。</li>
	</ol>
</li>
</ol>3. 抗凋亡活性测定
<ol><li>基因诱导细胞凋亡: 重复上述过程从2.1 到2.3。共染1µg 的 pDHsp/D-弹性蛋白/旗-他质粒 dna (含凋亡诱导基因) 与1µg 的质粒 dna [pDHsp70/V5-His 向量 (阴性对照), pDHsp70-Ac-P35/V5-His (阳性对照), pDHsp70-Ly-IAP3/V5-His, pDHsp70-Ly-IAP3-BIR/V5-His或 pDHsp70-Ly-IAP3-RING/V5-His, 分别.]。在5小时后热休克治疗, 进行细胞活力检测 (图 2)。</li>
	<li>化学诱导细胞凋亡: 重复上述步骤从2.1 到2.3。在步骤2.1.2 中, 板 1 x 106 sf9 单元格放入6井板中的每个井中。染4µg 质粒 DNA [pDHsp70/V5-His 矢量 (阴性对照)、pDHsp70-Ac-P35/V5-His (阳性对照)、pDHsp70-Ly-IAP3/V5-His、pDHsp70-Ly-IAP3-BIR/V5-His 或 pDHsp70-Ly-IAP3-RING/V5-His 分别.]。在5小时后热休克, 治疗 sf9 细胞2mL 的 ActD 细胞培养培养基16小时, 并进行细胞活力检测 (图 2)。 注意:6井板一井盖的最小容积为1毫升。</li>
	<li>执行上述抗凋亡活性试验, 包括3.1 和 3.2 triplicates。</li>
</ol>4. 细胞活性测定
<ol><li>通过添加1毫升 1x PBS 缓冲液, 1 分钟3次, 冲洗处理过的细胞。并用重悬细胞由吹打1毫升 1x PBS 缓冲器包含0.04% 台盼蓝和染色3分钟室温。将细胞悬浮液转移到1.5 毫升微细。 注意:通过混合9毫升 1x PBS 缓冲器和1毫升0.4% 台盼蓝溶液稀释0.4% 台盼蓝溶液。</li>
	<li>将10µL 台盼蓝染色细胞悬浮液转移到 hemocytometer, 用 P10 吸管, 并在光显微镜下计数存活完好的细胞。</li>
	<li>统计分析<ol>
<li>计算所记录的数据, 并以此作为 S.D. 所有计数的方法。</li>
		<li>使用学生的双尾 t 测试与 Microsoft Excel 一起分析数据。将统计重要数据定义为P值 < 0.05。</li>
	</ol>
</li>
</ol>

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作者声明他们没有竞争的财政利益。

基于热休克的 pDHsp/V5-His/sf9 细胞系统的概念在1994年的8中首先被克莱克et描述。杆状病毒基因启动子 (IE1) 和 果蝇hsp70 的比较表明 hsp70 在蚊子细胞10中的效率更高.此外, 由于热休克诱导, 在热休克治疗后, 可以精确控制蛋白质表达的时间。该系统随后应用于虾和 nucleopolyhedrovirus (NPV) 蛋白的蛋白质功能测定9,<sup cl...

两种蛋白表达系统通常用于生产蛋白质: 原核生物蛋白表达系统 (大肠杆菌基因表达系统) 和真核细胞蛋白表达系统.一种流行的真核细胞蛋白表达系统是杆状病毒表达载体系统 (BEVS)1。杆状首次被用作全球农业和森林害虫的生物控制剂。在过去的几十年中, 杆状被开发为蛋白质表达载体的生物技术工具。杆状的基因组由双链循环 DNA 和封装 nucleocapsids2组成。到目前为止, 已有七十八多个杆状病毒分离序列3。根据寄主昆虫细胞杆状病毒基因表达的时间级联, 基因转录可分为四个颞叶栅, 包括即刻早、延迟早、晚期和非常晚的基因 4.
BEVSs 被指定为非常晚基因促进者 (即, 多面体或 p10 启动子) 被用来驱动目标基因, 而重组杆状病毒是由同源重组产生的。重组杆状病毒在昆虫细胞中的外蛋白表达与转化后修饰的哺乳动物蛋白相似 (适合于糖蛋白的生成)。因此, 杆状病毒已被广泛使用5,6,7。然而, 一个限制是在昆虫细胞中存在着不同的 n-糖基化通路7。
因此, 建立了一种新的杆状病毒表达系统--瞬态基因表达系统。该系统在昆虫细胞中杆状病毒直接早期促进剂 (ie-1 启动子) 驱动下表达国外基因.通过使用该系统, 可以在ie-1启动子的控制下立即表达目标蛋白, 同时修改昆虫细胞中的 n-糖基化通路, 从而得到更好的 n-链寡糖7。此外, 杆状病毒直接早期基因由宿主细胞 RNA 聚合酶 II 转录, 不需要任何病毒因子激活 4.因此, 在短时间内, 外来蛋白可以在昆虫细胞中表达。目前, 瞬态基因表达系统是在杆状病毒体外细胞培养系统中进行蛋白质功能测定的重要技术之一。该系统可用于分析杆状病毒或外来蛋白质的功能。一个商业上可用的瞬态基因表达系统是基于直接早期基因 (IE) 促进者的Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) (OpIE2 和 OpIE1)。
然而, 当奥佩启动子的瞬态基因表达系统使用8,9,10时, 昆虫细胞外基因的表达水平仍然是一个问题。因此, 根据果蝇热休克蛋白 70 (hsp70) 基因8,9的启动子, 构建了另一个瞬时基因表达系统。在昆虫细胞的热休克诱导下, hsp70 的启动子比杆状病毒 IE 启动子更有效 10.在该系统中, 目标基因在果蝇热休克 70 (Dhsp70) 启动子的驱动下表达。利用 PCR 克隆方法可以很容易地将外来基因复制到瞬态基因表达质粒中。同时, 通过热休克诱导, 可以对基因表达的时机进行控制。
在本报告中, 我们遵循的方法和表达三不同截断的杆状病毒基因 (细胞凋亡3, iap3 从 毒蛾 MNPV) 使用热休克为基础的瞬态蛋白表达系统和进一步应用这些表达蛋白的抗凋亡活性分析。该系统既可以快速表达国外蛋白, 又可进一步应用于 sf9 细胞蛋白质抗凋亡活性的评价, 同时也具有应用于其他蛋白质活性测定的潜力。

<table><tbody><tr><td>Antibiotic-Antimycotic, 100X</td><td>Gibco</td><td>15240-062</td><td>for insect cell culture</td></tr><tr><td>Certified Foetal Bovine Serum</td><td>Bioind</td><td>04-001-1A</td><td></td></tr><tr><td>Sf-900 II SFM</td><td>Thermo Fisher</td><td>10902096</td><td>serum-free cell culture medium</td></tr><tr><td>Sf9 cells</td><td>ATCC</td><td>CRL-1711</td><td></td></tr><tr><td>25cm2 cell culture flask</td><td>Nunc, Thermo Fisher</td><td>156340</td><td></td></tr><tr><td>Inverted light microscopy</td><td>WHITED</td><td>WHITED WI-400</td><td></td></tr><tr><td>RBC HIT Competent Cell</td><td>Bioman</td><td>RH618-J80</td><td>&lt;em&gt;Escherichia coli&lt;/em&gt; (DH5&amp;alpha;)</td></tr><tr><td>L.B. Broth (Miller)</td><td>Bioman</td><td>LBL407</td><td></td></tr><tr><td>Agar, Bacteriological Grade</td><td>Bioman</td><td>AGR001</td><td></td></tr><tr><td>Zeocin</td><td>Invitrogen</td><td>ant-zn-1</td><td>selection antibiotic</td></tr><tr><td>PCR Master Mix (2X)</td><td>ThermoFisher</td><td>K0171</td><td></td></tr><tr><td>Geneaid Midi Plasmid Kit (Endotoxin Free)</td><td>Geneaid</td><td>PIE25</td><td></td></tr><tr><td>Actinomycin D</td><td>SIGMA</td><td>A9415</td><td></td></tr><tr><td>Corning 50 mL centrifuge tubes</td><td>SIGMA</td><td>CLS430829-500EA</td><td>50 mL tubes</td></tr><tr><td>Hemocytometer</td><td>Gizmo Supply Co</td><td>B-CNT-SLDE-V2</td><td></td></tr><tr><td>24-Well Multidish</td><td>Nunc, Thermo Fisher</td><td>142475</td><td>24-well plate</td></tr><tr><td>Cellfectin II Reagent</td><td>Thermo Fisher</td><td>10362100</td><td>cell transfectin reagent</td></tr><tr><td>PBS-Phosphate-Buffered Saline (10X) pH 7.4</td><td>Thermo Fisher</td><td>AM9624</td><td></td></tr><tr><td>4&amp;times;SDS Loading Dye</td><td>Bioman</td><td>P1001</td><td></td></tr><tr><td>Immobilon-P (PVDF Blotting Membranes)</td><td>Merck Milipore</td><td>IPVH00010</td><td>PVDF membranes</td></tr><tr><td>Mini Trans-Blot Cell system</td><td>BIO-RED</td><td>1703930</td><td>Blotting device</td></tr><tr><td>Ponceau S solution</td><td>SIGMA</td><td>6226-79-5</td><td></td></tr><tr><td>Anti-V5</td><td>SIGMA</td><td>V8137</td><td>rabbit anti-V5 antibody</td></tr><tr><td>Goat anti-rabbit IgG-horseradish peroxidase (HRP)</td><td>Jackson</td><td>111-035-003</td><td></td></tr><tr><td>Tween 20</td><td>Merck</td><td>817072</td><td></td></tr><tr><td>6-Well Multidish</td><td>Nunc, Thermo Fisher</td><td>145380</td><td></td></tr><tr><td>0.4 % trypan blue solution</td><td>AMRESCO</td><td>K940-100ML</td><td></td></tr><tr><td>P10 pipetman</td><td>Gilson</td><td>F144802</td><td></td></tr><tr><td>P1000 pipetman</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>Tape</td><td>Symbio</td><td>PPS7</td><td>24 well tape ( 19 mm&amp;times;36 M)</td></tr></tbody></table>

transient expression of foreign genes in insect cells (sf9) for protein functional assay

瞬态基因表达系统是在杆状病毒体外细胞培养系统中进行蛋白质功能分析的重要技术之一。该系统是在瞬态表达质粒中杆状病毒启动子控制下表达国外基因的一种方法。此外, 该系统可应用于杆状病毒本身或外来蛋白质的功能检测。以Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) 的直接早期基因启动子为基础, 建立了最广泛、最商业化的瞬时基因表达系统。然而, 在昆虫细胞中发现外来基因的表达水平较低。因此, 构建了一种用于改善蛋白质表达的瞬态基因表达系统。在该系统中, 构建了重组质粒, 以包含在果蝇热休克 70 (Dhsp70) 启动子控制下的目标序列。本协议介绍了这种基于热休克的 pDHsp/V5-His (V5 表位与6组氨酸)/斜纹 frugiperda细胞 (sf9 细胞) 系统的应用;该系统不仅用于基因表达, 而且可用于评价昆虫细胞中候选蛋白的抗凋亡活性。此外, 该系统可以转染一个重组质粒或联合转染两个潜在的功能拮抗重组粒的昆虫细胞。该协议证明了该系统的有效性, 并提供了该技术的实际应用实例。

基于热休克的 sf9斜纹夜蛾pDHsp/V5-His/细胞 (frugiperda 细胞) 系统, LyxyMNPV 中 Ly-IAP3 的全长和其他两个截断 (和环域) 抗原在 sf9 细胞中。pDHsp/V5-His 含有一个果蝇热休克蛋白启动子, 它通过使用细胞转录因子和翻译系统 (图 1)8 , 在42摄氏度的温度下驱动下游基因表达.,9,11。整...

Watch this Scientific Journal Video about Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay at JoVE.com

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

本协议描述了一种热休克诱导蛋白表达系统 (pDHsp/V5-His/sf9 细胞系统), 可用于表达外来蛋白或评价潜在外来蛋白的抗凋亡活性及其截断的氨基酸昆虫细胞中的酸。

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

transient-expression-foreign-genes-insect-cells-sf9-for-protein

Research

JoVE Journal

Genetics

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

12.2K 次观看.  National Ilan University. 本协议描述了一种热休克诱导蛋白表达系统 (pDHsp/V5-His/sf9 细胞系统), 可用于表达外来蛋白或评价潜在外来蛋白的抗凋亡活性及其截断的氨基酸昆虫细胞中的酸。

视频: Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

科研

遗传学

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Carboxylesterases 在抗杀虫剂家蝇、家蝇中的功能表征

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

生物化学

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 ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

生物工程

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. ...

生物学

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 ...

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

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

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

细菌，昆虫细胞和植物系统:重组蛋白表达在不同的生物工厂的比较分析

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

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

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

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

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

瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞

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