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>

<ol><li>Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Baculoviruses+as+gene+expression+vectors%5BTitle%5D)+AND+%22Annual+Reviews+in+Microbiology%22%5BJournal%5D)">Baculoviruses as gene expression vectors.</a> Annual Reviews in Microbiology. 42 (1), 177-199 (1988).</li>
<li>Theilmann, D. A., et al. Family Baculoviridae. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTheilmann%2C+DA+%22Virus+Taxonomy%3A+Eighth+Report+of+the+International+Committee+on+Taxonomy+of+Viruses%22">Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses</a>. Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., Ball, L. A. , Springer Press. New York. 1129-1185 (2005).</li>
<li>Nai, Y. S., Huang, Y. F., Chen, T. H., Chiu, K. P., Wang, C. H. Determination of nucleopolyhedrovirus' taxonomic position. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNai%2C+YS+%22Biological+Control+of+Pest+and+Vector+Insects%22">Biological Control of Pest and Vector Insects</a>. Shields, V. D. C. , InTech Press. Rijeka. 169-200 (2017).</li>
<li>Friesen, P. D. Regulation of baculovirus early gene expression. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFriesen%2C+PD+%22The+Baculoviruses%22">The Baculoviruses</a>. Miller, L. K. , Plenum Press. New York. 141-170 (1997).</li>
<li>Smith, G. E., Summers, M., Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+human+beta+interferon+in+insect+cells+infected+with+a+baculovirus+expression+vector%5BTitle%5D)+AND+%22Mol.+Cell.+Biol%22%5BJournal%5D)">Production of human beta interferon in insect cells infected with a baculovirus expression vector.</a> Mol. Cell. Biol. 3 (12), 2156-2165 (1983).</li>
<li>Luckow, V. A., Summers, M. D. <a target="_blank" href="https://www.nature.com/articles/nbt0188-47">Trends in the development of baculovirus expression vectors.</a> Nature Biotechnol. 6 (1), 47-55 (1988).</li>
<li>Jarvis, D. L., Finn, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modifying+the+insect+cell+N-glycosylation+pathway+with+immediate+early+baculovirus+expression+vectors%5BTitle%5D)+AND+%22Nature+Biotechnol%22%5BJournal%5D)">Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.</a> Nature Biotechnol. 14 (1996), 1288-1292 (1996).</li>
<li>Clem, R. J., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+programmed+cell+death+by+the+baculovirus+genes+p35+and+iap%5BTitle%5D)+AND+%22Mol.+Cell.+Biol%22%5BJournal%5D)">Control of programmed cell death by the baculovirus genes p35 and iap.</a> Mol. Cell. Biol. 14, 5212-5222 (1994).</li>
<li>Leu, J. H., Kuo, Y. C., Kou, G. H., Lo, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+cloning+and+characterization+of+an+inhibitor+of+apoptosis+protein+%28IAP%29+from+the+tiger+shrimp%2C+Penaeus+monodon%5BTitle%5D)+AND+%22Dev.+Comp.+Immunol%22%5BJournal%5D)">Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon.</a> Dev. Comp. Immunol. 32, 121-133 (2008).</li>
<li>Zhao, Y. G., Eggleston, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+promoters+for+transient+gene+expression+in+cultured+mosquito+cells%5BTitle%5D)+AND+%22Insect+Mol.+Biol%22%5BJournal%5D)">Comparative analysis of promoters for transient gene expression in cultured mosquito cells.</a> Insect Mol. Biol. 8 (1), 31-38 (1999).</li>
<li>Nai, Y. S., Yang, Y. T., Kim, J. S., Wu, C. Y., Chen, Y. W., Wang, C. H. <a target="_blank" href="https://link.springer.com/article/10.1007/s13355-016-0403-x">Baculoviral IAP2 and IAP3 encoded by Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) suppress insect cell apoptosis in a transient expression assay.</a> Appl. Entomol. Zool. 51, 305-316 (2016).</li>
<li>Geneaid™. Geneaid™ Midi Plasmid Kit & Geneaid™ Midi Plasmid Kit (Endotoxin Free) Instruction Manual Ver. 05.11.17. , Available from:
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<li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Western+Blotting%3A+Sample+Preparation+to+Detection%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Western Blotting: Sample Preparation to Detection.</a> J. Vis. Exp. (44), e2359(2010).</li>
<li>JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Separating Protein with SDS-PAGE. , Cambridge, MA. Available from:
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<li>Vucic, D., Kaiser, W. J., Harvey, A. J., Miller, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+reaper-induced+apoptosis+by+interaction+with+inhibitor+of+apoptosis+proteins+%28IAPs%29%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs).</a> Proc. Natl. Acad. Sci. USA. 94, 10183-10188 (1997).</li>
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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9,11。在本议定书中, 共使用了6种质粒结构: 三 (pDHsp/V5-His、pDHsp/Ac-p35/V5-His 和 pDHsp/D-弹性/旗-他) 是由宏列伊的9和其他三建筑 (pDHsp-iap3/V5-His、pDHsp-iap3-BIR/V5-His、和 pDHsp-iap3-RING/V5-His) 是由泥et al, 201611构建的。这似乎是昆虫细胞的启动子比杆状病毒基因促进者更有效的工作。但是, 在操作本协议时, 有几个要点需要考虑。在质粒转染前, 检查 DNA 序列的插入对基因表达有重要意义;因此, 它应该与 V5 表位融合, 形成一个 V5-tagged 融合蛋白在目标序列的末尾。此外, 不同的融合标记 (即, 标记表位) 也可以在基于 pDHsp 的体外蛋白质结合检测的向量中进行设计。此扩展过程将有助于进一步研究蛋白质-蛋白质相互作用9。因此, 商业 V5 多克隆抗体可用于蛋白质的检测。实验前应检测不同昆虫细胞的转染率。转染率较低可能导致无法检测到蛋白质表达;因此, 一个含有合适报告基因 (即, 绿色荧光基因) 的 pDHsp 载体可以转化为昆虫细胞, 以评估其转化效率。
这种热休克表达平台的主要局限性是蛋白质表达水平。在某些情况下, 发现低蛋白表达水平。根据一项试验研究, 当更多的质粒 DNA 转染成 sf9 细胞时, 没有明显的剂量依赖性效应 (未显示数据)。因此, 这种效应可能是由泛素蛋白酶体通路 (UPP) 或其他未知机制11引起的。研究人员应该在蛋白酶体抑制剂 (即, MG-132) 存在的情况下检查蛋白质表达, 以澄清和 recolve 此问题11。另一个限制是无法实现与重组病毒相关的蛋白质的可持续生产。因此, 蛋白质表达的稳定性和数量也是对该系统的关注。因此, 蛋白质生产的时间过程也应在功能测试前用西方印迹测定法进行检验。在该协议中, 我们比较了两个时间点 (1 和5小时的热休克), 并观察到目标蛋白生产的积累从1小时到5小时。为了确定蛋白质功能测定的最佳时机条件, 建议增加时间点。
在蛋白质功能检测中, 采用热休克瞬态表达系统, 表达了抗凋亡蛋白 Ac-P35 (阳性对照) 和凋亡诱导蛋白 (d-弹性蛋白酶)。这两种蛋白质被描述为功能拮抗剂9,11,15。因此, 向量/D-弹性环和 Ac-P35/D-RPR 可以作为一个负控制和积极控制, 分别。利用这种比较, 可以确定靶蛋白的抗凋亡活性。
该协议可为快速表达国外蛋白质提供平台, 也可进一步应用于昆虫细胞蛋白质抗凋亡活性的评价。一旦研究人员获得了蛋白质功能的初步结果, 该系统可用于进一步的研究。

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。整个技术流程图显示在图 2中。在1小时或5小时热休克后, 细胞裂解蛋白样缓冲, 并接受西方印迹检测, 以确认蛋白表达。结果表明, 全长蛋白、AC-P35、Ly-IAP3、Ly-IAP3-BIR 和 Ly-IAP3-RING 均可在1小时或5小时后热休克的转染细胞中检测到。此外, 在5小时后热休克中发现了蛋白质积累 (图 3)。因此, 根据数据, 蛋白质功能检测是在最大蛋白表达时间点 (5 小时后热休克) 进行的。
基因诱导的细胞凋亡和化学诱导的细胞凋亡可以适应这个系统来评估抗凋亡的活动 (图 2)。对于基因诱导的细胞凋亡, 两种构造 (凋亡诱导因子和靶基因质粒结构) 被共转染成 sf9 细胞, 然后热休克 activite 基因表达。同时, 采用热休克瞬态表达系统, 表达了抗凋亡蛋白 Ac-P35 (阳性对照) 和凋亡诱导蛋白 (d-弹性蛋白酶)。
热休克后, 两种基因开始表达并转化为蛋白质。阳性对照 (Ac-P35/D-RPR) 与载体/d-弹性细胞相比, 具有较高的抗凋亡活性, 达到了80% 的生存率。从其他构造的细胞活力结果, 研究人员可以比较抗凋亡活动彼此或积极控制 (图 4A)。对于化学诱导的细胞凋亡, 只有一个构造被转染成 sf9 细胞。在5小时后热休克后, 添加了化学物质 (ActD), 在 16 h (图 2) 后测量细胞的活力。与正控制 (AC-P35) 或负控制 (矢量) 相比, 每个构造显示了对抗凋亡活动的各种影响 (图 4B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56693/56693fig1.jpg"> 图 1: 基于 pDHsp/V5-His 向量的ly-iap3相对质粒结构的图和核苷酸序列。起始密码子 (ATG) 以粗体显示。下划线表示限制酶切割点 (后 III 在红色颜色;BamHI以蓝色为颜色);绿色序列表示 Dhsp70 启动子区域。<a href="//cloudflare.jove.com/files/ftp_upload/56693/56693fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56693/56693fig2.jpg"> 图 2: 热休克诱导蛋白表达系统 (pDHsp/V5-His/sf9 细胞系统) 的流程图.以基因诱导细胞凋亡和化学诱导细胞凋亡治疗为起始步骤 (与凋亡诱导基因质粒的共转染) 或热休克 (化学诱导细胞凋亡) 后一步, 分别用于抗凋亡试验。修改后的图形和图例已通过 Springer11的权限复制。<a href="//cloudflare.jove.com/files/ftp_upload/56693/56693fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56693/56693fig3.jpg"> 图 3: 使用热休克诱导蛋白表达系统 (pDHsp/V5-His/sf9 细胞系统) AC-P35、Ly-IAP3、Ly-IAP3-BIR 和 Ly-IAP3-RING 的过度表达.在1小时或5小时后的热休克, 细胞裂解物的收获, 并接受了西方印迹化验和 SDS/页。(A) 以α V5 抗体和 (B) 为荷载控制的考马斯蓝染色的西方印迹检测。<a href="//cloudflare.jove.com/files/ftp_upload/56693/56693fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56693/56693fig4.jpg"> 图 4: 对d-弹性细胞或放线菌 D 诱导的凋亡的可行性测定。() sf9 细胞以 pDHsp70/V5-His 矢量 (仅限矢量) 转染, 并与 pDHsp70/V5-His 向量、pDHsp70/Ac-P35/V5-His、pDHsp70/Ly-IAP3/V5-His、pDHsp70/Ly-IAP3-BIR/V5-His、pDHsp70/一起转染 pDHsp70/drpr/FLAG-HisLy-IAP3-RING/V5-His, 分别。向量与 P35、向量与 Ly-IAP3 之间的差异具有统计学意义 (t检验;P < 0.05)。(B) sf9 细胞以 pDHsp70/V5-His 向量或 pDHsp70/Ac-P35/V5-His、pDHsp70/Ly-IAP3/V5-His、pDHsp70/Ly-IAP3-BIR/V5-His、pDHsp70/Ly-IAP3-RING/V5-His 分别转染, 热休克后5小时, 添加 ActD, 16 小时后,测定细胞活力。向量与 P35 的差异具有统计学意义 (t检验;P < 0.05)。修改后的图形和图例已通过 Springer11的权限复制。<a href="//cloudflare.jove.com/files/ftp_upload/56693/56693fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>名称</td>
			<td>序列</td>
		</tr>
<tr>
<td>pDHsp-IAP3-HindIII-F</td>
			<td>5´-GGAAGCTTACCATGGACGACGAACGACGCAG 3´</td>
		</tr>
<tr>
<td>pDHsp-IAP3-BamHI-R</td>
			<td>5´-GCGGATCCCGGATGTAGGAACACCTTGA 3´</td>
		</tr>
<tr>
<td>iap3-BIR-BamHI-r</td>
			<td>5´-CGGGATCCCGGCAGATCGCCGCCGCGGA 3´</td>
		</tr>
<tr>
<td>iap3-RING-HindIII-F</td>
			<td>5´-GGAAGCTTACCGAGCTCATAAAAAGGCCCGT-3´</td>
		</tr>
<tr>
<td>pDhsp70-F2</td>
			<td>5´-CTGCAACTACTGAAATCAACCAAG-3´</td>
		</tr>
<tr>
<td>Op-IE2R</td>
			<td>5´-GACAATACAAACTAAGATTTAGTCAG-3´</td>
		</tr>
</tbody></table>表 1: 用于基于 PCR 的克隆方法的底漆集. *下划线的脱氧核糖核酸基地对表明了制约酵素站点。修改后的底漆列表是通过 Springer11的许可复制的。

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

Nanyang Technological University

Research

JoVE Journal

Genetics

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

12.3K 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

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

甲病毒转导系统：可视化，以免蚊虫感染的工具

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

科研

免疫与感染

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 ...

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 ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. PRMTs can either mono- or dimethylate arginine guanidino groups symmetrically or asymmetrically. The enzymology of these proteins is a complex and intensely investigated area that requires milligram quantities of high-quality recombinant protein. The baculovirus expression vector system (BEVS) employing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera frugiperda 9 (Sf9) insect cells has been used for expression screening and production of many PRMTs, including PRMT 1, 2, and 4 through 9. To simultaneously screen for the expression of multiple constructs of these proteins, including domains and truncated fragments as well as the full-length proteins, we have applied scalable methods utilizing adjustable and programmable multichannel pipettes, combined with 24- and 96-well plates and blocks. Overall, these method adjustments enabled a large-scale generation of bacmid DNA, recombinant viruses, and protein expression screening. Using culture vessels with a high-fill volume of Sf9 cell suspension helped to overcome space limitations in the production pipeline for single batch large-scale protein production. Here, we describe detailed protocols for the efficient and cost-effective expression of functional PRMTs for biochemical, biophysical, and structural studies.

The baculovirus expression vector system (BEVS) is a robust platform for expression screening and production of protein arginine methyltransferases (PRMTs) to be used for biochemical, biophysical, and structural studies. Milligram quantities of material can be produced for the majority of PRMTs and other proteins of interest requiring a eukaryotic expression platform.

使用巴库洛病毒表达载体系统生产重组PRMT蛋白

Protein arginine methyltransferases (PRMTs) methylate arginine residues on a wide variety of proteins that play roles in numerous cellular processes. ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

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 ...

生物学

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 ...