这项工作得到了来自北卡罗来纳州立大学 Guozhou 许的新的教员启动基金的支持。

注: 采用修饰表达载体的昆虫细胞/杆状病毒系统, 用于分泌植物蛋白的表达和结晶。
1. 用 GP67 信号肽对杆状病毒表达载体进行植物蛋白分泌表达的改进
<ol><li>合成一个 DNA 片段, 包含 5 ' BglII 切割站点13, GP67 分泌信号序列, 和一个多克隆网站与 NotI, BamHI, EcoRI, StuI, 贝里沙, SpeI, XbaI, PstI 和 XhoI (图 1A)。 注: 由于 BglII 和 BamHI 消化 dna 导致相同的粘合剂末端, 线性化的载体可以结扎到被消化的脱氧核糖核酸片断, 当退火的结扎序列的 BglII 和 BamHI 站点将被变异到不是裂解的序列无论是 BglII 或 BamHI14。</li>
	<li>消化4µg 的 dna 与 BglII 和 XhoI, 和4µg 的表达载体 dna BamHI 和 XhoI14。将每种限制酶的10个单位与1x 浓度反应缓冲器中的 DNA 混合 (50 毫米醋酸钾, 20 毫米三醋酸盐, 10 毫米醋酸镁, 100 µg/毫升 BSA, pH 7.9 在25°c) 和孵化混合物在37°c 为 4 h。<ol>
<li>用 dna 凝胶萃取试剂盒15纯化分离的 dna 片段和线性化的载体 dna。</li>
	</ol>
</li>
	<li>在含有 1x T4 DNA 连接酶反应缓冲器 (50 毫米三盐酸) 的 10-µL 结扎反应混合物中, 将100的线性化向量 DNA 与 400 ng 的被消化 dna 片段混合在一起; 10 毫米氯化镁2, 1 毫米 ATP, 和10毫米的德勤, pH 7.5 在25°c) 和5个单位的 T4 DNA连接酶.孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL 的 DH5α合格细胞, 遵循标准的转换协议, 并选择在氨苄西林-LB 琼脂板16上产生的菌落。拿起 5-10 殖民地和生长每殖民地在2毫升的 LB 培养基包含100µg/毫升氨苄西林在 220 rpm 和37°c 在一个震动孵化器为 16 h。</li>
	<li>提取每个质粒 dna 与 dna miniprep 试剂盒17和确认克隆的 dna 测序与底漆 AGTATTTTACTGTTTTCGTAACAG。</li>
	<li>PCR-放大a.拟南芥基因片段编码残留物 30-642 从合成 TDR 基因构造 (向前底漆: CATG GCGGCCGC CTCAAGTTTTCACCTCAACTCTTGTC, 反向底漆: GC GGATCC CTA GTG GTG ATG GTG GTG GTG ACCGTCTATATCTGCATTTCCGGC)。在 dna 聚合酶反应缓冲器中放大30周期的 dna, 其中包含0.5 毫米 dNTP 混合, 0.2 µM 底漆, 0.1 µg 的模板 dna, 0.4 毫米氯化镁2和1反应单元的 dna 聚合酶。<ol>
<li>为 PCR 周期步骤, 设置最初变性在95°c 三十年代, 然后30周期变性在95°c 三十年代, 退火在55°c 为三十年代, 并且引伸在72°c 为 1 min, 以最后的引伸在72°c 为5分钟。</li>
	</ol>
</li>
	<li>用 NotI 和 BamHI 限制限制性酶对 PCR 片段和修饰载体进行消化。用线性化向量结扎基因片段。将 100 ng 的线性化矢量 DNA 与400的消化 dna 片段 (含 T4 dna 连接酶反应缓冲器和5单位 T4 dna 连接酶) 的µL 结扎反应混合物混合。孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL 的 DH5α合格细胞, 遵循标准的转换协议, 并选择在氨苄西林-LB 琼脂板16上产生的菌落。通过 DNA 测序确认正确的克隆。</li>
</ol>2. 昆虫 Hemolin 信号肽用于植物蛋白分泌表达的杆状病毒表达载体的改进
<ol><li>合成一个 DNA 片段, 包含 5 ' BglII 切割站点13, 昆虫 hemolin 分泌信号序列, 和一个多克隆网站与 NotI, BamHI, EcoRI, StuI, 贝里沙, SpeI, XbaI, PstI 和 XhoI (图 1B)。</li>
	<li>消化4µg 的 dna 与 BglII 和 XhoI, 和4µg 的表达载体 dna BamHI 和 XhoI14。将每种限制酶的10个单位与1x 浓度反应缓冲器中的 DNA 混合 (50 毫米醋酸钾, 20 毫米三醋酸盐, 10 毫米醋酸镁, 100 µg/毫升 BSA, pH 7.9 在25°c) 和孵化混合物在37°c 为 4 h。<ol>
<li>用 dna 凝胶萃取试剂盒15纯化分离的 dna 片段和线性化的载体 dna。</li>
	</ol>
</li>
	<li>在含有 1x T4 DNA 连接酶反应缓冲器 (50 毫米三盐酸) 的 10-µL 结扎反应混合物中, 将100的线性化向量 DNA 与 400 ng 的被消化 dna 片段混合在一起; 10 毫米氯化镁2, 1 毫米 ATP, 和10毫米的德勤, pH 7.5 在25°c) 和5个单位的 T4 DNA连接酶.孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL DH5α合格细胞, 并选择氨苄西林-LB 琼脂板上的菌落。拾起 5-10 殖民地并且生长每个殖民地在一个2毫升 LB 媒介包含100µg/毫升氨苄西林在 220 rpm 和37°c 在一个震动孵化器为 16 h。</li>
	<li>用 dna miniprep 试剂盒17提取质粒 dna, 并通过 AGTATTTTACTGTTTTCGTAACAG 的 dna 测序确认克隆。</li>
	<li>PCR-放大a. 芥花粉受体激酶 3 (PRK3) 基因片段编码残留物 20-237 从合成 PRK3 基因构造 (向前底漆: CATG GCGGCCGC CTACAAAACGTTAGTGAATCGGAACC, 反向底漆: CGC GGATCC CTA GTG GTG ATG GTG GTG GTGTGAAGGTTTCTCATCGCATTCTATATTC)。在 dna 聚合酶反应缓冲器中放大30周期的 dna, 其中包含0.5 毫米 dNTP 混合, 0.2 µM 底漆, 0.1 µg 的模板 dna, 0.4 毫米氯化镁2和1反应单元的 dna 聚合酶。<ol>
<li>为 PCR 周期步骤, 设置初始变性在95°c 三十年代, 然后30周期变性在95°c 三十年代, 退火在55°c 为三十年代, 并且延长在72°c 为三十年代在每个周期之内, 并且最后的引伸在72°c 为5分钟。</li>
	</ol>
</li>
	<li>用 NotI 和 BamHI 限制限制性酶对 PCR 片段和修饰载体进行消化。用线性化向量结扎基因片段。将 100 ng 的线性化矢量 DNA 与400的消化 dna 片段 (含 T4 dna 连接酶反应缓冲器和5单位 T4 dna 连接酶) 的µL 结扎反应混合物混合。孵育反应混合物在16°c 为16小时。</li>
	<li>将结扎合剂的5µL 转化为100µL 的 DH5α合格细胞, 遵循标准的转换协议, 并选择在氨苄西林-LB 琼脂板16上产生的菌落。通过 DNA 测序确认正确的克隆。</li>
</ol>3. 以重组蛋白表达盒为载体的杆状病毒结构的制作和扩增
<ol><li>利用构造质粒100的 DH10Bac, 在杆状病毒表达系统1的协议下, 转化40µL 的合格细胞。在37摄氏度的48小时内孵化转化板。</li>
	<li>从每个变换板上提取两个白色的菌落, 将每个菌落接种2毫升的 LB 培养基, 其中含有50µg 卡那霉素、7µg/毫升庆大霉素, 以及10µg/毫升四环素。遵循杆状病毒表达系统1的协议提取和验证 bacmid DNA。整除每个 DNA 在两个管与20µL 每一个, 并存储管在-20 摄氏度。 注意: 以下步骤需要无菌操作。</li>
	<li>在培养基中培养10% 胎牛血清 (血清) 和100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素, 在27摄氏度至80% 井板的 Sf9 细胞。根据组织培养板上覆盖面积的百分比估计汇流的百分比。</li>
	<li>在两个无菌1.5 毫升离心管中准备以下解决方案。<ol>
<li>管 1: 每次转染, 稀释20µL bacmid DNA 成100µL unsupplemented Sf9 细胞培养基, 无抗生素。轻轻地把稀释后的管子的侧面混合。</li>
		<li>管 2: 对每转染, 稀释8µL 脂转染试剂成100µL unsupplemented Sf9 细胞培养基无抗生素。将稀释液彻底混合, 吹打上下6x。</li>
	</ol>
</li>
	<li>结合两个解决方案, 轻轻地混合在一起, 吹打慢慢向上和向下 3x, 并孵化他们在室温下 20-40 分钟。</li>
	<li>用3毫升的 unsupplemented Sf9 细胞培养培养基 Sf9 单层细胞 2x, 无抗生素。每次转染, 添加0.8 毫升的 unsupplemented Sf9 细胞培养基, 不含抗生素, 每管含有脂质 DNA 混合物。轻轻地将管子的内容混合在一起, 慢慢地吹打3x。从盘子中吸取洗涤介质, 然后用转染混合物覆盖样品。</li>
	<li>在添加转染混合物后, 在27摄氏度的5小时内孵化出所染的板。用完整的 Sf9 细胞培养基取代转染培养基, 其中含有10% 只血清和100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素。在27摄氏度孵育转染板 4 d。</li>
	<li>收获和放大重组杆状病毒。<ol>
<li>在 4 d 孵化后, 在不育的15毫升圆锥管中收集感染板的上清。以 1010 x g为5分钟旋转上清, 去除细胞碎片。丢弃颗粒并将上清醒酒到干净的管子上, 将其贮存在摄氏4摄氏度, 长达1年。 注意: 这是 P0 病毒。它可以 aliquoted 和储存在-80 摄氏度, 在较长的时间内。</li>
		<li>为了放大每种重组病毒, 在150毫米的板块上生长一层 Sf9 细胞到80% 汇合。从盘子中吸取培养基, 将2毫升的 P0 病毒添加到附着在盘子上的细胞中, 在27摄氏度孵化器中孵化出1小时的平板。每隔15分钟摇动盘子, 将培养基与细胞混合。<ol>
<li>添加25毫升完整的恩典的媒体含有10% 血清和100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素。在27摄氏度孵化器中孵化出 3 d 的盘子, 以获得 P1 通道病毒。</li>
		</ol>
</li>
		<li>在不育的50毫升圆锥管中收集被感染的盘子的上清, 并在 1010 x g处旋转5分钟以去除细胞碎片。醒酒到干净的管, 并保存在4摄氏度, 长达1年。 注: P1 病毒可以 aliquoted 和储存在-80 摄氏度, 较长一段时间。</li>
		<li>为了将病毒从 P1 扩大到 P2 通道, 在150毫米的盘子上长出一层 Sf9 细胞到90% 汇合处, 吸出盘子的培养基, 在盘子中加入2毫升 P1 病毒, 并在27摄氏度孵化器中孵化1小时。</li>
		<li>重复步骤3.8.2 和3.8.3 获取病毒的 P2 和 P3 通道。不要将 P3 病毒存储2月以上。</li>
	</ol>
</li>
</ol>4. 蛋白质表达, NiNTA 纯化和结晶
<ol><li>培养 1 L Hi5 昆虫细胞在文化媒介与100µg 或 ml (或100国际单位/毫升) 青霉素-链霉素抗生素到密度 2 x 106在 100 rpm 在27°c 孵化器振动筛。向下旋转 570 x g的细胞为5分钟, 醒酒上清, 并用重悬的细胞在20毫升的新鲜生产的 P3 病毒, 并保持在室温下1小时. 轻轻摇动旋转瓶, 并用重悬细胞1x 每15分钟。</li>
	<li>将细胞转移到1升的培养基上, 用100µg/毫升 (或100国际单位/毫升) 青霉素-链霉素抗生素, 培养细胞在 100 rpm 在27°c 孵化器振动筛为 72 h. 在 1010 x g的细胞中旋转5分钟, 然后收集上清。</li>
	<li>使用 ph 指示条将上清液的 ph 值调整为 8.0, 加入0.1 米 HCl 或0.1 米氢氧化钠, 并在最终浓度下添加捆绑缓冲器, 在 pH 8.0、100毫米氯化钠和5毫米咪唑中含有20毫米的三个缓冲器。添加一定数量的 NiNTA 树脂 (10-40 毫升) 根据估计产量的表达他标记的重组蛋白。<ol>
<li>在4°c 的1小时内, 将溶液与低速的磁搅拌混合在一起, 清洗树脂并洗脱标准 NiNTA 纯化协议18中的绑定蛋白。</li>
	</ol>
</li>
	<li>以纯化蛋白为离子交换和凝胶过滤层析, 以及其他蛋白质纯化方法, 产生均匀样品。 注: 对于 TDR 胞外区, 20 毫米三, pH 8, 100 毫米氯化钠被用作凝胶过滤缓冲器。对 PRK3 胞外区, 20 毫米双三, pH 6, 100 毫米氯化钠作为首选凝胶过滤缓冲剂。</li>
</ol>

<ol>
	<li>Contreras-Gomez, A., Sanchez-Miron, A., Garcia-Camacho, F., Molina-Grima, E., Chisti, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+using+the+baculovirus-insect+cell+expression+system.">Protein production using the baculovirus-insect cell expression system.</a> Biotechnology Progress. 30, 1-18 (2014).</li><li>Khan, S., Ng, M. L., Tan, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+severe+acute+respiratory+syndrome+coronavirus+3a+protein+and+the+assembly+of+coronavirus-like+particles+in+the+baculovirus+expression+system.">Expression of the severe acute respiratory syndrome coronavirus 3a protein and the assembly of coronavirus-like particles in the baculovirus expression system.</a> Methods in Molecular Biology. 379, 35-50 (2007).</li><li>Possee, R. D., King, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+Transfer+Vectors.">Baculovirus Transfer Vectors.</a> Methods in Molecular Biology. 1350, 51-71 (2016).</li><li>Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., McCawley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+two+cell+lines+from+the+insect+Spodoptera+frugiperda+(Lepidoptera;+Noctuidae).">The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae).</a> In Vitro. 13, 213-217 (1977).</li><li>Hink, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Established+insect+cell+line+from+the+cabbage+looper,+Trichoplusia+ni.">Established insect cell line from the cabbage looper, Trichoplusia ni.</a> Nature. 226, 466-467 (1970).</li><li>Davis, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+recombinant+protein+production+of+eight+insect+cell+lines.">Comparative recombinant protein production of eight insect cell lines.</a> In Vitro Cellular &amp; Development Biology - Animal. 29A, 388-390 (1993).</li><li>Wickham, T. J., Nemerow, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+growth+methods+and+recombinant+protein+production+in+BTI-Tn-5B1-4+insect+cells+using+the+baculovirus+expression+system.">Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system.</a> Biotechnology Progress. 9, 25-30 (1993).</li><li>Davis, T. R., Trotter, K. M., Granados, R. R., Wood, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+of+alkaline+phosphatase+as+a+reporter+gene+for+evaluation+of+production,+glycosylation+and+secretion.">Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion.</a> Nature Biotechnology. 10, 1148-1150 (1992).</li><li>Murphy, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression,+secretion,+and+large-scale+purification+of+recombinant+HIV-1+gp120+in+insect+cell+using+the+baculovirus+egt+and+p67+signal+peptides.">Enhanced expression, secretion, and large-scale purification of recombinant HIV-1 gp120 in insect cell using the baculovirus egt and p67 signal peptides.</a> Protein Expression and Purification. 4, 349-357 (1993).</li><li>Sun, S. C., Lindstrom, I., Boman, H. G., Faye, I., Schmidt, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolin:+an+insect-immune+protein+belonging+to+the+immunoglobulin+superfamily.">Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily.</a> Science. 250, 1729-1732 (1990).</li><li>Li, Z., Chakraborty, S., Xu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+CLE+peptide+perception+by+plant+receptors+implicated+from+structural+and+functional+analyses+of+TDIF-TDR+interactions.">Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions.</a> PLoS One. 12, e0175317 (2017).</li><li>Chakraborty, S., Pan, H., Tang, Q., Woolard, C., Xu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Extracellular+Domain+of+Pollen+Receptor+Kinase+3+is+structurally+similar+to+the+SERK+family+of+co-receptors.">The Extracellular Domain of Pollen Receptor Kinase 3 is structurally similar to the SERK family of co-receptors.</a> Scientific Reports. 8, 2796 (2018).</li><li>Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+of+a+gene.">Total synthesis of a gene.</a> Science. 203, 614-625 (1979).</li><li>Bahl, C. P., Marians, K. J., Wu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+method+for+inserting+specific+DNA+sequences+into+cloning+vehicles.">A general method for inserting specific DNA sequences into cloning vehicles.</a> Gene. 1, 81-92 (1976).</li><li>Xu, W., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Vaccine-Derived+Polioviruses,+Relevance+for+the+Global+Polio+Eradication+Initiative.">Isolation and Characterization of Vaccine-Derived Polioviruses, Relevance for the Global Polio Eradication Initiative.</a> Methods in Molecular Biology. 1387, 213-226 (2016).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> Journal of Molecular Biology. 166, 557-580 (1983).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+plasmid+DNA+from+bacterial+colonies+using+the+QIAGEN+Miniprep+Kit.">Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit.</a> Journal of Visualized Experiments. (6), 247 (2007).</li><li>Reddy, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+the+alpha+subunit+of+the+epithelial+sodium+channel,+ENaC.">Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC.</a> Protein Expression and Purification. 117, 67-75 (2016).</li><li>Harris, T. J., Emtage, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+heterologous+genes+in+E.+coli.">Expression of heterologous genes in E. coli.</a> Microbiological Sciences. 3, 28-31 (1986).</li><li>Kaur, J., Kumar, A., Kaur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+optimization+of+heterologous+protein+expression+in+E.+coli:+Roadblocks+and+reinforcements.">Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements.</a> International Journal of BIological Macromolecules. 106, 803-822 (2018).</li><li>Guo, Y., Yang, F., Tang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+of+Protein+Secretion+in+Yeast+and+Animal+Cells.">An Overview of Protein Secretion in Yeast and Animal Cells.</a> Methods in Molecular Biology. 1662, 1-17 (2017).</li><li>Blight, M. A., Chervaux, C., Holland, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+pathway+in+Escherichia+coli.">Protein secretion pathway in Escherichia coli.</a> Current Opinion in Biotechnology. 5, 468-474 (1994).</li><li>Idiris, A., Tohda, H., Kumagai, H., Takegawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+protein+secretion+in+yeast:+strategies+and+impact+on+protein+production.">Engineering of protein secretion in yeast: strategies and impact on protein production.</a> Applied Microbiology and Biotechnology. 86, 403-417 (2010).</li><li>Wormald, M. R., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoproteins:+glycan+presentation+and+protein-fold+stability.">Glycoproteins: glycan presentation and protein-fold stability.</a> Structure. 7, R155-R160 (1999).</li><li>Geisler, C., Mabashi-Asazuma, H., Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+and+History+of+Glyco-Engineering+in+Insect+Expression+Systems.">An Overview and History of Glyco-Engineering in Insect Expression Systems.</a> Methods in Molecular Biology. 1321, 131-152 (2015).</li><li>Li, Z., Chakraborty, S., Xu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+crystallographic+studies+of+the+extracellular+domain+of+the+first+plant+ATP+receptor,+DORN1,+and+the+orthologous+protein+from+Camelina+sativa.">X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa.</a> Acta Crystallographica Section F: Structural BIology and Crystallization Communications. 72, 782-787 (2016).</li><li>Aricescu, A. R., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+recombinant+glycoproteins+in+mammalian+cells:+towards+an+integrative+approach+to+structural+biology.">Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology.</a> Current Opinion in Structural Biology. 23, 345-356 (2013).</li><li>Reuter, L. J., Bailey, M. J., Joensuu, J. J., Ritala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-up+of+hydrophobin-assisted+recombinant+protein+production+in+tobacco+BY-2+suspension+cells.">Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells.</a> Plant Biotechnology Journal. 12, 402-410 (2014).</li><li>Dohi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+virus-mediated+expression+of+a+foreign+protein+in+suspension-cultured+plant+cells.">Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells.</a> Archives of Virology. 151, 1075-1084 (2006).</li></ol>

作者没有什么可透露的。

鉴于生物系统中数以千计的蛋白质的大小和稳定性的多样性, 研究实验室通常有经验来决定哪些异种表达系统必须选择用于表达特定的蛋白质。大肠杆菌表达系统往往是由于细菌生命周期短、培养基成本低、相对容易扩展19而导致蛋白质表达的首选。然而, 对于大小超过 60 kDa 的大真核蛋白的表达, 使用大肠杆菌系统往往导致不溶性蛋白在包涵体或聚集20。对于这些困难的蛋白质表达, 以及其他分泌蛋白, 杆状病毒-昆虫细胞表达系统可能比大肠杆菌更有利。特别是在表达分泌真核蛋白时, 这种改良的杆状病毒-昆虫细胞表达系统是首选的选择。
许多分泌真核蛋白, 包括植物分泌蛋白, 需要复杂的糖基化, 二硫化形成, 和其他转化后的修饰, 蛋白质折叠和分泌21。大肠杆菌系统没有细胞机械来处理许多真核蛋白22所需的复杂修改。酵母具有相对较先进的糖基化系统, 比大肠杆菌23。然而, 对于高真核生物和植物中分泌蛋白的表达, 杆状病毒-昆虫细胞系统具有重要的优势。由于糖基化影响蛋白质折叠和稳定性24, 许多分泌的重组蛋白表达在大肠杆菌和酵母系统有低产量和倾向于聚集, 大概是由于不正确的蛋白质折叠。对杆状病毒-昆虫系统进行了改进, 以改善蛋白质糖基化, 使其成为分泌真核蛋白25表达的理想系统。在本研究中, GP67 和 hemolin 信号肽已成功地用于指导两种植物受体蛋白在蛋白质结晶中的分泌表达。然而, 有了这些研究, 无论是信号肽的选择是一个关键的步骤, 它需要更系统的比较研究与一套目标基因从不同的有机体。
利用 GP67 和 hemolin 信号肽提高蛋白分泌表达的思想, 是由两种基因的高表达率驱动的。然而, 如果表达宿主的蛋白分泌物和翻译后修饰机械被表达的重组蛋白超载, 则分泌的重组蛋白可能没有足够的修饰, 特别是糖基化。两种改良的载体都被用来成功地表达了许多植物分泌蛋白11,12,26, 其中许多往往聚集, 这可能是由于不正确或不充分的糖基化。因此, 对于这些难以表达的蛋白质, 必须对强弱信号肽序列进行测试, 并对表达的重组蛋白的质量进行比较。请记住, 弱信号肽序列将降低蛋白质的整体产量;然而, 它可以给出分泌机械的表达宿主足够的能力来处理蛋白质的分泌和修饰。
除了在昆虫细胞中表达外源分泌蛋白外, 哺乳动物细胞和植物细胞表达系统已成功地应用于重组哺乳动物和植物蛋白的过度表达, 分别为27, 28,29。与异源系统相比, 哺乳动物或植物分泌蛋白质的近内源性表达条件将使宿主细胞中的几乎本机修饰机制产生良好的折叠蛋白。使用近本机表达系统的告诫是, 所表达的重组蛋白可能干扰细胞的生理功能, 可能对蛋白质最终表达率产生不利影响。

研究实验室有能力生产大量的均质重组蛋白, 用于生化和生物物理特征, 尤其是 x 射线晶体学研究。大肠杆菌、酵母、昆虫细胞、哺乳动物细胞、植物细胞等具有良好的外源表达系统, 其中杆状病毒介导的昆虫细胞表达系统是其中最常用的技术生产大量的结构折叠大型重组真核蛋白的蛋白质结晶1。
对杆状病毒表达系统的表达载体进行了设计, 以包含强多角体或 P10 启动子, 以产生高产量的重组细胞内蛋白2,3。为了制作重组杆状病毒, 该基因被克隆成含有杆状使用杆状多 nucleopolyhedroviral 基因组多角体 (polh) 轨迹的昆虫载体。然后对生成的构造进行排序, 验证其正确的开放阅读框架 (ORF)。通过转染过程将正确的构造引入宿主昆虫细胞。兴趣基因通过同源重组插入病毒基因组。这一事件导致重组病毒基因组的生产, 然后复制, 以产生重组发芽病毒粒子1。
在表达系统中最常用的昆虫细胞是 Sf9 和高五 (Hi5) 细胞。Sf9 细胞是 Sf21 蛹卵巢细胞的克隆分离物, Hi5 细胞是由父母夜蛾镍卵巢细胞系 TN-3684,5所衍生的克隆分离株。transfections、病毒扩增和斑块检测是在 Sf9 细胞上进行的, 而 Hi5 细胞通常被选择以产生更高数量的重组蛋白6。值得注意的是, Hi5 细胞由于其产生突变病毒的倾向而不适合于病毒后代的产生和扩增。传统上, 25-30 °c 的温度范围被认为对昆虫细胞的培养是有益的。然而, 据报道, 27-28 °c 是最佳的温度为昆虫细胞生长和传染7,8。
在基因之前引入一个强烈的信号序列是分泌蛋白的高表达所必需的。该信号序列将有效地引导转化后的重组蛋白转化为内质网的蛋白质分泌和转化后的修改必要的适当折叠和稳定3。信号肽序列, 如杆状病毒包络蛋白 GP64/67, 蜜蜂蜂毒等, 已被选择, 以促进分泌重组蛋白的表达在杆状病毒介导的表达系统3。GP67 的信号肽的引入, 以提高分泌重组蛋白的表达率, 与使用目标基因9的内在信号肽相比较。Hemolin 是大丝蛾Hyalophora cecropia的血淋巴蛋白, 引起细菌感染10。由于诱导表达水平较高, 该基因的信号肽序列可以用来调节杆状病毒-昆虫细胞系统中重组蛋白的分泌表达。
Tracheary 元素分化抑制因子受体 (TDR) 和花粉受体激酶 3 (PRK3) 都属于植物亮氨酸丰富的重复受体样激酶 (LRR RLK) 蛋白11,12 的家庭.为了研究植物受体蛋白家族的结构和功能, 以及促进其他植物分泌蛋白的结构和生化特性, 对杆状病毒-昆虫细胞表达系统进行了改进, 以提高蛋白质质量和产量。利用两种修饰表达载体在杆状病毒-昆虫细胞表达系统中成功地表达了 TDR 和 PRK3 的胞外域。TDR 和 PRK3 蛋白的胞外域均已结晶。本文报道了两种改进的杆状病毒表达载体的大量重组分泌植物蛋白的表达和纯化, 通过在启动子和多克隆之间加入 GP67 或 hemolin 信号序列。网站。

<table>
	<tbody>
		<tr>
			<td>Incubator shaker</td>
			<td>VWR</td>
			<td>Model Excella E25</td>
			<td>27 oC</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model 2005</td>
			<td>27 oC</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>BECKMAN</td>
			<td>Model J-6</td>
			<td>with a swing bucket rotor</td>
		</tr>
		<tr>
			<td>Herculase II Fusion DNA Polymerase</td>
			<td>Agilent</td>
			<td>600677-51</td>
			<td>For PCR amplification of DNA</td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>BIO-RAD</td>
			<td>Model C1000 Touch</td>
			<td>For PCR amplification of DNA</td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Model I 24</td>
			<td>For growing baceria culture</td>
		</tr>
		<tr>
			<td>Customer DNA synthesis</td>
			<td>GENSCRIPT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI (HF)</td>
			<td>New England Biolabs</td>
			<td>R3136S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>New England Biolabs</td>
			<td>R0144S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>NotI (HF)</td>
			<td>New England Biolabs</td>
			<td>R3189S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td>DNA ligation</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>DNA purification from Agorase gel</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>Plasmid DNA purification from bacteria culture</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>15510-019</td>
			<td>For DNA gel electropherosis</td>
		</tr>
		<tr>
			<td>MAX Efficiency DH5&#945; competen cell</td>
			<td>Invitrogen</td>
			<td>18-258-012</td>
			<td>For transformation of DNA ligation mixture</td>
		</tr>
		<tr>
			<td>Lennox L LB Broth</td>
			<td>Research Product International Corp.</td>
			<td>L24066-5000.0</td>
			<td>For making bacteria culture</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Thermo Fisher Scientific</td>
			<td>11593-019</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>15160-054</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Thermo Fisher Scientific</td>
			<td>64-75-5</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15710-064</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>MAX Efficiency DH10Bac competent cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>10361-012</td>
			<td>For making bacmid DNA</td>
		</tr>
		<tr>
			<td>S.O.C. Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>15544-034</td>
			<td>For DNA transformation</td>
		</tr>
		<tr>
			<td>CellFECTIN II Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362-101</td>
			<td>Insect cell transfection reagent</td>
		</tr>
		<tr>
			<td>Bac-to-Bac Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>10359-016</td>
			<td>Baculovirus-insect cells expression kit</td>
		</tr>
		<tr>
			<td>Bluo-gal</td>
			<td>Thermo Fisher Scientific</td>
			<td>15519-028</td>
			<td>For isolation of recominant Bacmid DNA</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Fisher Scientific</td>
			<td>15529-019</td>
			<td>For isolation of recominant Bacmid DNA</td>
		</tr>
		<tr>
			<td>pFastBac1</td>
			<td>Thermo Fisher Scientific</td>
			<td>10360014</td>
			<td>Baculorirus expression vector</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>11496015</td>
			<td>Sf9 monolayer cells</td>
		</tr>
		<tr>
			<td>Hi5 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>B85502</td>
			<td>High Five insect cells</td>
		</tr>
		<tr>
			<td>Grace&#8217;s insect medium, unsupplemented</td>
			<td>Thermo Fisher Scientific</td>
			<td>11595030</td>
			<td>Sf9 cell transfection minimum medium</td>
		</tr>
		<tr>
			<td>Grace&#8217;s insect medium, supplemented</td>
			<td>Thermo Fisher Scientific</td>
			<td>11605102</td>
			<td>Sf9 monolayer cell culture complete medium</td>
		</tr>
		<tr>
			<td>Sf-900 II SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10902104</td>
			<td>Sf9 suspension cell culture medium without FBS</td>
		</tr>
		<tr>
			<td>Express Five SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10486025</td>
			<td>Hi5 cell culture medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 ml (10,000 I.U./ml)</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>FBS Certified</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-772-1B</td>
			<td>Flat-bottom</td>
		</tr>
		<tr>
			<td>150 mm plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>353025</td>
			<td>100/case</td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1415-2500</td>
			<td>500 tubes/bag</td>
		</tr>
		<tr>
			<td>15 ml conical screw cap centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1475-0511</td>
			<td>25 tubes/bag</td>
		</tr>
		<tr>
			<td>50 ml conical screw cap centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1500-1211</td>
			<td>25 tubes/bag</td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td>NiNTA resin</td>
		</tr>
		<tr>
			<td>pH-indicator strips</td>
			<td>EMD Millipore Corporation</td>
			<td>1.09535.0001</td>
			<td>pH 0 - 14</td>
		</tr>
	</tbody>
</table>

modifying baculovirus expression vectors to produce secreted plant proteins in insect cells

对科学家来说, 表达重组分泌真核蛋白用于结构和生物化学研究是一个挑战。杆状病毒介导的昆虫细胞表达系统是用于表达重组真核分泌蛋白的系统之一, 具有一定的转化后修饰。分泌蛋白需要通过分泌通路进行蛋白质糖基化、二硫化键形成和其他转化后的修饰。为改善现有分泌植物蛋白的昆虫细胞表达, 在启动子和多克隆点之间添加 GP67 或 hemolin 信号肽序列, 对杆状病毒表达载体进行修改。新设计的改进后的载体系统成功地生产出了拟南芥可溶性重组分泌植物受体蛋白的高产。对两种表达的植物蛋白, 拟南芥和 PRK3 膜受体的胞外域进行了结晶, 用于 x 射线晶体的研究。改进后的矢量系统是一种改良的工具, 可用于动物王国中重组分泌蛋白的表达。

如图 1所示, 两种改进的 pFastBac1 杆状病毒表达载体用于表达 GP67 或 hemolin 信号序列的分泌蛋白, 以取代目标基因的内在信号序列。病毒 GP67 和昆虫 hemolin 基因已被证明具有较高的分泌表达水平的细胞。融合蛋白与这两个信号序列中的任何一个有望大大提高分泌表达水平。在这两种改进的向量中, 都设计了相同的多个克隆站点 (MCS)。NotI 站点被蓄意放置在 MCS 的最 5 ' 一侧, 因为 NotI 有一个八核苷酸序列, 而不是许多其他限制站点中最常见的六核苷酸序列。因此, 比起其他限制点, NotI 站点在目标基因中较少存在, 使得限制消化和大多数目标基因的克隆比使用其他限制点更可行。克隆的目标基因之间的 NotI 和下游限制点将只引入三氨基酸残留物, GGR, 之前的目标基因。
pBac1-GP67 和 pBac1-Hem 已成功地用于表达A. 南芥受体 TDR 和 PRK3 的胞外域 (图 2)。利用工程 C-端 6-组氨酸标记对重组蛋白进行纯化后, Hi5 细胞的平均蛋白质产量约为20毫克/升 NiNTA。每种蛋白质的纯度接近或高于 50%, 用 SDS 页和考马斯染色法进行检查。
用尺寸排除色谱法进一步纯化了 TDR 和 PRK3 胞外域的重组蛋白 (图 2)。纯化后的蛋白质浓缩为5毫克/毫升, 然后进行结晶筛选。条件, 产生了每种蛋白质的初步晶体, 经过优化, 直到蛋白质晶体的大小大于20µm 的观察 (图 3)。两种蛋白质的晶体结构报告了11,12。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58283/58283fig1.jpg"> 图 1: 修改后的 pFastBac1 向量的映射.在这些面板中显示的 DNA 序列入 pFastBac1 载体的多角体启动子的下游 , 以拥有目标基因的信号肽序列和多个克隆点 ( MCS ) 。两个向量都包含相同的 MCS。(A) 本小组在 pBac1-GP67 向量中显示了多角体启动子下游的克隆站点序列。翻译的 GP67 信号肽氨基酸序列以红色着色。MCS 中的每个唯一限制站点都带下划线, 上面标记的站点名称。(B) 本小组在 pBac1-Hem 向量中显示了多角体启动子下游的克隆站点序列。翻译的 hemolin 信号肽氨基酸序列以红色着色。MCS 中的每个唯一限制站点都带下划线, 上面标记的站点名称。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58283/58283fig1large.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/58283/58283fig2.jpg"> 图 2: 在杆状病毒-昆虫细胞系统中, 修饰 pFastBac1 载体的蛋白表达.(A) 在 Hi5 昆虫细胞中表达了 TDR 的胞外区, 用镍亲和性和粒度排除色谱法纯化, 并在 SDS 页凝胶上解决。用含有5毫米咪唑 (洗涤 1) 的缓冲器清洗一次镍树脂, 第二次用20毫米咪唑 (洗涤 2)。(B) 这是一个 SDS 页分析, 显示了表达和镍亲和纯化 (NiNTA), 以及 PRK3 胞外区的大小排除色谱。标有相应尺寸的分子量 (兆瓦) 标记。每个面板中的红色箭头表示重组 TDR 和 PRK3 胞外区蛋白的表达和预期大小。表达蛋白的多波段性质可能是由于异构糖基化所致。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58283/58283fig2large.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/58283/58283fig3.jpg"> 图 3:拟南芥TDR 和 PRK3 胞外域的蛋白质晶体.(A) 本小组显示了一个. 南芥TDR 的胞外域的蛋白质晶体。(B) 这个小组显示了 PRK3 的胞外域的蛋白质晶体。每个图片下面显示一个刻度条。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58283/58283fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells at JoVE.com

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins 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

在这里, 我们提出了利用昆虫细胞和杆状病毒蛋白表达系统来产生大量植物分泌蛋白的蛋白质结晶的协议。用 GP67 或昆虫 hemolin 信号肽对昆虫细胞中植物蛋白分泌表达进行了改进, 并对杆状病毒表达载体进行了修正。

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

modifying-baculovirus-expression-vectors-to-produce-secreted-plant

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.4K 次观看.  North Carolina State University. 在这里, 我们提出了利用昆虫细胞和杆状病毒蛋白表达系统来产生大量植物分泌蛋白的蛋白质结晶的协议。用 GP67 或昆虫 hemolin 信号肽对昆虫细胞中植物蛋白分泌表达进行了改进, 并对杆状病毒表达载体进行了修正。

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

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

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

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

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

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

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

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce.
Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.

Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.

如何定量光活化荧光蛋白在大容量和活细胞中的分数

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

通过基因表达分析的改良方法从谷物种子获取高质量的转录数据

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.