Name: a comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems
Uploaded: 2015-03-23T14:00:00.000+00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems at JoVE.com

This work was supported by the COST action &#8216;Molecular pharming: Plants as a production platform for high-value proteins&#8217; FA0804. The Authors thank Dr Anatoli Giritch and Prof. Yuri Gleba for providing the MagnICON vectors for research purposes.

1.构建表达载体<ol><li>商业重组克隆系统: <ol><li>扩增全长的靶基因（hGAD65mut）用合适的引物，允许在该基因的5&#39;末端加入一个CACC夹具如前所述2的序列。 </li><li> 1的摩尔比插入物:载体和1μl的通过组装在6微升的总体积的反应中，使用1.5克隆凝胶纯化的扩增产物，根据定向克隆试剂盒的规格，在输入向量（拓扑异构酶结合的）盐溶液（0.2M NaCl和0.01的MgCl 2）。孵育在室温下5分钟（RT）。 </li><li>变换整个反应进化学感受态根据定向克隆试剂盒的规格和屏幕获得的菌落通过菌落PCR 3，使用M13正向和反向引物的大肠杆菌细胞包括在定向克隆试剂盒。从PO隔离质粒根据质粒DNA制备试剂盒的规格和序列插入用M13正向和反向引物，以确保不存在突变3的sitive菌落。 </li><li>使用所获得的进入克隆通过拉姆达重组酶克隆酶II酶（LR）与特定的目标矢量进行重组反应:为在细菌细胞的重组蛋白质的表达与pDEST17（产生pDEST17.G65mut），在植物中（瞬时或稳定）与pK7WG2 （产生pK7WG2.G65mut），在杆状病毒/昆虫细胞系统与线性化病毒DNA（产生Baculo.G65mut）4。组装的反应中，根据克隆酶试剂盒的规格，用100ng入门载体，150纳克目标矢量和2μl酶混合物中的10微升的最终体积。孵育混合物在RT搅拌1小时。 </li><li>改造重组产物进化学感受态根据克隆酶试剂盒规格大肠杆菌细胞。通过PCR 3的屏幕获得的菌落</SUP&gt;使用对于所有的转化反应得到的菌落在同一反向GAD65mut特异引物（5&#39;-CACACGCCGGCAGCAGGT-3&#39;）和以下的具体目的向量的正向引物:用于pDEST17:5&#39;-TAATACGACTCACTATAGGG-3&#39;;对于pK7WG2:5&#39;-AAGATGCCTCTGCCGACAGT-3&#39;;对于杆状病毒线性载体:5&#39;-AAATGATAACCATCTCGC-3&#39;。 </li><li>使用商用的DNA小量制备试剂盒分离质粒DNA并确认目标序列的存在通过PCR 3，使用在先前步骤中所描述的相同的特定的引物组合。 </li><li>使用所得PCR阳性质粒转化不同靶生物，取决于如在以下步骤中所述选择用于重组蛋白质表达的平台上。 </li></ol></li><li> MagnICON系统5-7: <ol><li>克隆靶基因的3&#39;-模块pICH31070，如先前详细描述的7，得到pICH31070.G65mut。使用以下具体反应周期:30分钟在37℃，5分钟，在50℃，然后用5分钟在80℃。 </li></ol></li></ol> 2.重组蛋白表达<ol><li>细菌细胞系统: <ol><li>转换成pDEST17.G65mut E.大肠杆菌 BL21（DE3）用标准技术和筛选菌落生长在含有氨苄青霉素（100微克/毫升）的LB培养基，通过菌落PCR，使用下列转基因特异性引电感受态细胞:5&#39;-CTGGTGCCAAGTGGCTCAGA-3&#39;和5&#39; -CACACGCCGGCAGCAGGT-3&#39;，用58℃的退火温度和20秒的延伸时间。执行与在管的塑料尖端溶解细菌细胞后在20微升的总体积PCR反应。 </li><li>在3ml含氨苄青霉素（100微克/毫升）的LB培养基过夜接种单个菌落，在37℃。 </li><li>稀释的细菌培养1:100在100毫升的新鲜LB培养基中（含ampicill在），并孵育在37℃下进行1-6小时，直到最终的OD 600为0.8。 </li><li>通过加入isopropil-β-D-1-tiogalattopiranoside（IPTG）中的1mM的最终浓度诱导重组蛋白质表达并孵育培养在37℃下剧烈振荡3小时。 </li><li>通过离心收集细胞，在4000×g离心20分钟，并用细菌沉淀用于蛋白质提取（步骤3.1.1.1）。 </li></ol></li><li>杆状病毒/昆虫细胞系统: <ol><li>种子草地夜蛾 （的Sf9）细胞到6孔板（8×10 5个细胞每孔），并用2ml无补充Grace昆虫培养基洗两次。 </li><li>卸下并更换介质逐滴加入转染混合物（5微升LR重组反应，6微升Celfectin液和200微升非补充Grace的昆虫培养基）。 </li><li>孵育板在27℃5小时。 </li><li>删除并用2ml新鲜SF-9替换转染混合物00培养基，补充有10％胎牛血清，10μg/ ml的庆大霉素和100μM更昔洛韦选择重组杆状病毒的克隆。 </li><li>培养96小时，在27°C后，收集介质（V1病毒原料），离心机在4000 XG到4℃，除去细胞和大的杂物和存储在黑暗中。 </li><li>种子1×10 6个Sf9昆虫细胞每井在2.5ml SF-900含有10％胎牛血清，10μg/ ml的庆大霉素和100μM更昔洛韦和感染在每100微升V1股票的良好介质。 </li><li>孵育细胞3天，在27℃。 </li><li>收集并在4000×g的离心机中。 </li><li>存储上清液（V2高滴度的股票），在4℃。 </li><li>种子1×10 6个Sf9昆虫细胞每井在2.5ml SF-900含有10％胎牛血清，10μg/ ml的庆大霉素和100μM更昔洛韦和感染用25微升V2库存中每一井平台。 </li><li>孵育细胞3天，在27℃。 </li><li>收集的细胞通过离心机在4000×g离心，用昆虫细胞沉淀用于蛋白质提取（步骤3.1.2。1）。 </li></ol></li><li> 本氏烟草瞬时表达系统: <ol><li>成长N.本生植物在温室中，在自然光中的温度范围18-23℃。对于农杆菌感染使用4-5周龄的植物。 </li><li>保持在一个气候室agroinfected植物在22℃，13小时昼/ 11小时夜的光周期。 </li><li>商业重组克隆系统: <ol><li>引入pK7WG2.G65mut在根癌农杆菌菌株EHA105电感受态细胞如前所述8和筛选菌落，2后生长在LB培养基中含有利福平（50微克/毫升），链霉素（300微克/毫升）和壮观霉素（100微克/毫升）使用下列转基因特异的引物温育天在28℃，通过菌落PCR 8:5&#39;-CATGGTGGAGCACGACACGCT-3＆＃8217;和5&#39;-CACACGCCGGCAGCAGGT-3&#39;，用58℃的退火温度和50秒的延伸时间。进行在20微升的总体积PCR反应。 </li><li>在28℃接种细菌在30ml的LB培养基中含有利福平（50微克/毫升），链霉素（300微克/毫升）和壮观霉素（100微克/毫升）两天的。 </li><li>收集细菌离心4000 xg离心20分钟，重悬沉淀在10ml浸润缓冲液（10mM MES，10mM的MgCl 2的，100μM的乙酰丁香酮，pH值5.6）。测量的OD 600值，然后将其稀释在相同的缓冲液调节到0.9。 注意:需要的浸润3 N.总体积本生植物为30毫升</li><li>使用2.5ml的针注射器渗入土壤杆菌悬浮液中N.叶本生 。小心缓慢地注入叶板与从注射器的悬浮液中。打入3扩充升单株屋檐和使用三个工厂为一式三份。 注:为健康和安全原因穿在渗透过程保护眼睛。 </li><li>收集农杆菌渗入叶后2天感染（dpi）的并冷冻在液氮中。商店的植物组织在-80℃下。 </li></ol></li><li> MagnICON系统: <ol><li>引入MagnICON矢量- 5&#39;模块（pICH20111），3&#39;模块（pICH31070.G65mut），以及整合模块（pICH14011） -中A.农杆菌使用标准技术GV3101菌株。筛选菌落，生长在含LB培养基50微克/毫升利福平和使用特异性引物对每个矢量适当的载体中特异性的抗生素（50μg/ ml的羧苄青霉素用于pICH20111和pICH14011，50微克/毫升卡那霉素用于pICH31070），通过菌落PCR。 </li><li>分别接种三A.癌农杆菌菌株在5ml LB培养基中含有50微克/毫升利福平和适当的载体特异性抗生素和动摇一夜之间在28°C。 </li><li>粒料过夜细菌培养物通过离心在4000×g离心20分钟，并重新悬浮在它们两卷的10mM MES（pH为5.5）和10mM 硫酸镁的初始细菌培养物。 </li><li>混合含有三个矢量细菌悬浮液的等体积，并使用该悬浮液的混合为N的注射器浸润叶本生 。渗透每个三厂展开的叶片。 </li><li>收集农杆菌渗入叶在4 dpi和冷冻在液氮中。 </li><li>商店的植物组织在-80℃下。 </li></ol></li></ol></li><li> 烟草稳定表达系统: <ol><li>成长和维护N.的组培苗烟草 （ 变种SR1。）在固体植物培养基（4.4 g / L的Murashige和Skoog -质谱-介质包括维生素，30g / L的蔗糖，pH值5.8，第7克/升的植物琼脂）在受控条件下在气候室中于25 ℃，16小时/ 8小时白天/ NIGHT制度。 </li><li>启动A的预培养农杆菌 EHA105含有该表达载体pK7WG2.G65mut在5ml用合适的抗生素的液体的YEB培养基（利福平50微克/毫升，链霉素300微克/毫升，大观霉素100微克/毫升），并在定轨振荡器组生长过夜，在28℃下在200rpm。 </li><li>使用预培养1毫升接种在YEB培养基加上抗生素50ml的农杆菌培养物（利福平50微克/毫升，链霉素300微克/毫升，大观霉素100微克/毫升），并生长24小时，直到所述细菌培养是饱和（OD 600值0.5〜1.0）。 </li><li>收集细菌离心4000 xg离心20分钟，重悬沉淀在50ml液体植物培养基。重复此步骤两次，以彻底去除抗生素。 </li><li>就拿第一健康完全展开的叶片排出体外烟草植物和切成大约为1平方厘米。 </li><li>转让叶件到深培养皿含有细菌悬液并在黑暗中保留20分钟。 </li><li>起停牌删除叶片，吸干在无菌滤纸上。 </li><li>放置叶片与正面侧（上叶表面）上的固体植物培养基含有1.0微克/毫升6-苄氨基嘌呤（BAP）和0.1微克/毫升萘乙酸（NAA）和孵育板两天在气候室中在受控条件（25℃，16小时日/ 8小时夜间）。 </li><li>转印叶片上的固体培养基（含1.0微克/毫升的BAP，0.1微克/毫升的NAA，500微克/ ml的头孢噻肟，100微克/毫升卡那霉素）与背面（叶的下表面）中与介质接触。 </li><li>孵育平板2-3周，在气候室中于25℃下用16小时/ 8小时白天/黑夜制度以诱导枝条形成。继代培养，每2周通过转移叶植食固体植物培养基含有1.0微克/毫升BAP，0.1微克/毫升NAA，500微克/ ml的头孢噻肟和100微克/毫升卡那霉素。 </li><li>当枝条似乎它们转移到含有固体培养基含500微克/ ml的头孢噻肟和100微克/毫升卡那霉素以诱导根的形成Magenta盒。孵育小植株以16小时昼/ 8小时夜晚光周期在25℃下进行1-2周。 </li><li>当根形式，在温室中的植物转移到土壤中。 </li><li>收集一块叶每个工厂和分离基因组DNA与商业套件。 </li><li>检测转基因通过特异性PCR的存在。使用下列转基因特异性引物使用30纳克基因组DNA为模板进行PCR扩增的:5&#39;-CTGGTGCCAAGTGGCTCAGA-3&#39;和5&#39;-CACACGCCGGCAGCAGGT-3&#39;，用58℃的退火温度和20秒的延伸时间。进行在50微升的总体积PCR反应。 </li><li>在Tris-乙酸-EDTA（TAE）缓冲液的1％琼脂糖凝胶（40毫Tr的分析通过电泳的220碱基对产物是，20毫乙酸，和1mM EDTA）。 </li><li>选择含有靶基因的转基因植物。 </li><li>收集一个叶片从每个选定的转基因植物，并立即冷冻在液氮中。 </li><li>制备总蛋白提取物从植物的叶组织（步骤3.1.3），并如先前所述2来选择最佳的重组蛋白表达的烟草植物用western印迹分析测试每个样品。 </li><li>选择表现最好的植物袋鲜花盛开，防止交叉授粉前。 </li><li>开花，果实成熟和种子干燥后，收集袋。 </li><li>从谷壳分离的种子，并将其储存在干燥的房间，在控制温度（20-24℃）。 </li><li>播下干燥种子，以产生第二代转基因植物，并随后选择性能最佳的植物是自花传粉。 </li><li>重复在步骤2.4.19-2.4.21用于后续世代中描述的相同的过程。 </li></OL&gt; </li></ol> 3，重组蛋白表达的分析<ol><li>总蛋白的提取: <ol><li>细菌细胞: <ol><li>如在步骤2.1.5所述，在Tris缓冲盐水的一半培养体积重悬细菌细胞沉淀，收集（TBS - 2毫摩尔Tris /盐酸，500mM的NaCl）中pH 7.4的补充有1mM的phenylmethanesulfonylfluoride（PMSF）。 </li><li>声处理重悬细胞沉淀三次40秒以一半的功率，同时保持样品在冰上。 </li><li>澄清的溶胞产物离心以14000×g离心20分钟，在4℃。 </li><li>转移上清液至一个干净的管中并在-80℃下单独储存既上清液和沉淀。 </li><li>溶解包涵体，收集在沉淀，在TBS pH为8.0，在室温下剧烈振荡培养过夜补充有6M尿素的一半细胞裂解物体积。 </li><li>离心以10,000 xg离心在室温下25分钟，并在一个干净的收集上清管。 </li><li>同时存储上清，沉淀在-80℃。 </li></ol></li><li>昆虫细胞: <ol><li>洗感染的昆虫细胞沉淀，如在步骤2.2.12中描述，用1ml磷酸盐缓冲盐水的收集（PBS - 137 mM氯化钠，2.7 mM的氯化钾，10毫的Na 2 HPO 4，1.8毫KH 2 PO 4）pH为7.4。 </li><li>重悬的细胞在200μl的裂解缓冲液（20mM的Tris /盐酸pH为8.0，0.5M氯化钠，3mM的β巯基乙醇和1％吐温20）和在冰上孵育30分钟。 </li><li>离心机溶解细胞以14000×g离心20分钟，在4℃。 </li><li>收集可溶性部分并储存在-80℃并弃沉淀。 </li></ol></li><li>植物叶片组织: <ol><li>研磨N.本生或N.使用研钵和杵烟草组织以细粉末在液氮中。 </li><li>均质在300微升提取缓冲地面组织的100毫克（40毫摩尔的Hepes pH值7.9，5mM的DTT，1.5％CHAPS），增刊emented用3微升的蛋白酶抑制剂和解冻的冰。 注意:植物组织重量（克）之间的选择比来缓冲体积（ml）为1:3。 </li><li>离心提取液以30,000×g离心30分钟，在4℃。 </li><li>在一个干净的管中并储存在-80℃下收集上清液。 </li></ol></li></ol></li><li>考马斯亮蓝染色凝胶: <ol><li>制备适当稀释的总蛋白提取物（ 例如，2微升的植物提取物，5微升的细菌和昆虫提取物），以10微升的提取缓冲液的终体积，并添加5微升的3×样品缓冲液（1.5M的Tris /盐酸pH值6.8，3％SDS，15％甘油，4％β巯基乙醇）至最终浓度1倍。 </li><li>煮沸样品10分钟。 </li><li>分离的蛋白通过10％SDS-PAGE。 </li><li>电泳后，在考马斯溶液A（0.05％亮蓝R-250，25％异丙醇，10％乙酸）中在微波炉中存在大约两分钟热度凝胶s直至沸点。 </li><li>冷却凝胶至室温下轻轻摇动。 </li><li>弃考马斯亮蓝染色溶液A. </li><li>通过加热在考马斯溶液B存在脱色凝胶（0.05％亮蓝R-250，25％异丙醇），C（0.002％亮蓝R-250，10％乙酸）和D（10％乙酸）每次按照相同的协议进行染色。 </li><li>最后加热步骤与溶液D后，留下凝胶脱色直到背景清晰9。 </li></ol></li><li> Western blot分析: <ol><li>电泳后，转移在室温下分离使用标准技术，用4％的乳块的蛋白质到硝酸纤维素膜在PBS pH 7.4的1小时。 </li><li>过夜孵育印迹在4℃或4小时，在室温下，在含有1针对靶蛋白的兔一抗封闭溶液:10,000和补充有0.1％Tween-20的。 </li><li>经过一抗标签ING，用封闭溶液含有0.1％Tween-20洗涤膜3次，每次5分钟。 </li><li>孵育与辣根过氧化物酶（HRP）偶联物的抗兔抗体以1:10,000 1.5小时。 </li><li>洗膜为每个用PBS-T 5分钟5次（PBS中补充有0.1％吐温20）。 </li><li>过程中使用的化学发光过氧化物酶底物免疫印迹。 </li></ol></li><li>放射免疫测定法（RIA）: <ol><li>允许试剂（抗血清，示踪和蛋白A琼脂糖）达到室温和吸管20微升蛋白提取物样品和标准在不同浓度的（从市售的重组hGAD65的300-2,400纳克/毫升）成12×75mm的聚苯乙烯管中。 </li><li>加入20微升抗血清，准备稀释1:30从T1D病人的血浆，血清。 </li><li>添加50μl的示踪剂（125-I-GAD65）的孵育2小时，在室温下进行。设置与示踪剂的管只以估计总活性。 </li><li>加入50微升蛋白A琼脂糖和孵育1小时，在室温下进行。 </li><li>分配1ml冷的PBS缓冲液到每个管中，并离心以1500 xg离心在4℃下进行30分钟。 </li><li>倒出管除去上清液和吸收残留的液体上轻轻拍打管吸水纸。 </li><li>测量通过在γ计数器计数1分钟免疫沉淀的放射活性在所有管中。 </li><li>剧情在数比例与总活性校准的结合率（B / T％），建立标准曲线，和读取样品浓度GAD关闭校准曲线10。 注:限制区域应该被设计用于存储，处理和处置的放射性物质。 </li></ol></li></ol>

<ol>
	<li>Hampe, C. S., Hammerle, L. P., Falorni, A., Robertson, J., Lernmark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+of+K396R+of+the+65+kDa+glutamic+acid+decarboxylase+active+site+obliterates+enzyme+activity+but+not+antibody+binding.">Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding.</a> FEBS Lett. 488 (3), 185-189 (2001).</li><li>Avesani, L., 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=Recombinant+human+GAD65+accumulates+to+high+levels+in+transgenic+tobacco+plants+when+expressed+as+an+enzymatically+inactive+mutant.">Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant.</a> Plant Biotechnol. J. 9 (8), 862-872 (2010).</li><li>Sambrook, J., 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=.">.</a> Molecular Cloning: A laboratory manual. Second Edition. , (1989).</li><li>Avesani, L., 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+analysis+of+different+biofactories+for+the+production+of+a+major+diabetes+autoantigen.">Comparative analysis of different biofactories for the production of a major diabetes autoantigen.</a> Transgenic Res. 23, 281-291 (2014).</li><li>Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V., Gleba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+planta+engineering+of+viral+RNA+replicons:+efficient+assembly+by+recombination+of+DNA+modules+delivered+by+Agrobacterium.">In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium.</a> Proc. Natl. Acad. Sci. (USA). 101 (18), 6852-6857 (2004).</li><li>Gleba, Y., Klimyuk, V., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+the+expression+of+proteins+in+plants).">Viral vectors for the expression of proteins in plants).</a> Curr. Opin. Biotechnol. 18, 134-141 (2007).</li><li>Engler, C., Kandzia, R., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one+pot,+one+step,+precision+cloning+method+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PLoS One. 3 (11), (2008).</li><li>Xu, R., Li, Q. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+streamline+cloning+of+genes+into+binary+vectors+in+Agrobacterium+via+the+Gateway+TOPO+vector+system.">Protocol: streamline cloning of genes into binary vectors in Agrobacterium via the Gateway TOPO vector system.</a> Plant Methods. 4 (4), 1-7 (2008).</li><li>Fairbanks, G., Steck, T. L., Wallach, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+analysis+of+the+major+polypeptides+of+the+human+erythrocyte+membrane.">Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.</a> Biochemistry. 10 (13), 2606-2617 (1971).</li><li>Falorni, A., 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=Radioimmunoassay+detects+the+frequent+occurrence+of+autoantibodies+to+the+Mr+65,000+isoform+of+glutamic+acid+decarboxylase+in+Japanese+insulin-dependent+diabetes.">Radioimmunoassay detects the frequent occurrence of autoantibodies to the Mr 65,000 isoform of glutamic acid decarboxylase in Japanese insulin-dependent diabetes.</a> Autoimmunity. 19, 113-125 (1994).</li><li>Hunt, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gene+to+protein:+a+review+of+new+and+enabling+technologies+for+multi-parallel+protein+expression.">From gene to protein: a review of new and enabling technologies for multi-parallel protein expression.</a> Protein Expr. Purif. 40 (1), 1-22 (2005).</li><li>Arzola, L., 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=Transient+co-expression+of+post-transcriptional+silencing+suppressor+for+increased+in+planta+expression+of+a+recombinant+anthrax+receptor+fusion+protein.">Transient co-expression of post-transcriptional silencing suppressor for increased in planta expression of a recombinant anthrax receptor fusion protein.</a> Int. J. Mol. Sci. 12 (8), 4975-4990 (2011).</li><li>Merlin, M., Gecchele, E., Capaldi, S., Pezzotti, M., Avesani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+recombinant+protein+production+in+different+biofactories:+the+green+perspective.">Comparative evaluation of recombinant protein production in different biofactories: the green perspective.</a> Biomed. Res. Int. 2014, 136419 (2014).</li><li>Avesani, L., 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=Improved+in+planta+expression+of+the+human+islet+autoantigen+glutamic+acid+decarboxylase+(GAD65).">Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65).</a> Transgenic Res. 12 (2), 203-212 (2003).</li><li>Leuzinger, 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=Efficient+agroinfiltration+of+Plants+for+high-level+transient+expression+of+recombinant+proteins.">Efficient agroinfiltration of Plants for high-level transient expression of recombinant proteins.</a> J Vis Exp. (77), (2013).</li></ol>

The authors declare that there is no conflict of interests regarding the publication of this paper.

在这项研究中三个不同的平台上进行了比较重组人蛋白的表达:细菌细胞，杆状病毒/昆虫细胞和植物。 （ - MagnICON和pK7WG2基础-稳定的即瞬态）的植物为基础的平台是通过利用三个广泛使用的表达技术的进一步探索。选择用于该实验中，hGAD65mut，靶蛋白已在不同的系统13先前已经表示，它的生产和功能是很容易检测和可测量14。 细菌细胞不是一种有效的生...

Recombinant proteins are commercially mass-produced in heterologous expression systems with the aid of emerging biotechnology tools. Key factors that have to be considered when choosing the heterologous expression system include: protein quality, functionality, process speed, yield and cost.
In the recombinant protein field, the market for pharmaceuticals is expanding rapidly and, consequently, most biopharmaceuticals produced today are recombinant. Proteins can be expressed in cell cultures of bacteria, yeasts, molds, mammals, plants and insects, as well as in plant systems (either via stable- or transient-transformation) and transgenic animals; each expression system has its inherent advantages and limitations and for each target recombinant protein the optimal production system has to be carefully evaluated.
Plant-based platforms are arising as an important alternative to traditional fermenter-based systems for safe and cost-effective recombinant protein production. Although downstream processing costs are comparable to those of microbial and mammalian cells, the lower up-front investment required for commercial production in plants and the potential economy of scale, provided by cultivation over large areas, are key advantages.
We evaluated plants as bioreactors for the expression of the 65 kDa isoform of human glutamic acid decarboxylase (hGAD65), one of the major autoantigen in Type 1 autoimmune diabetes (T1D). hGAD65 is largely adopted as a marker, both for classifying and monitoring the progression of the disease and its role in T1D prevention is currently under investigation in clinical trials. If these trials are successful, the global demand for recombinant hGAD65 will increase dramatically.
Here, we focus on the expression of the enzymatically inactive counterpart of hGAD65, hGAD65mut, a mutant generated by substituting the lysine residue that binds the cofactor PLP (pyridoxal-5'-phosphate) with an arginine residue (K396R)1. 
hGAD65mut retains its immunogenicity and, in plant and insect cells, accumulates up to ten-fold higher than hGAD65, its wild-type counterpart. It was hypothesized that the enzymatic activity of hGAD65 interferes with plant cell metabolism to such an extent that it suppresses its own synthesis, whereas hGAD65mut, the enzymatically-inactive form, can be accumulated in plant cells to higher levels.
For the expression of hGAD65mut, the use of different technologies, widely used in plant biotechnology, was explored here and compared to traditional expression platforms (Escherichia coli and Baculovirus/insect cell-based).
In this work, the recombinant platforms developed for the expression of hGAD65mut comprising traditional and plant-based systems were reviewed and compared on the basis of process speed and yield, and of final product quality and functionality.

<table><tbody><tr><td>Yeast extract</td><td>Sigma</td><td>Y1333</td><td></td></tr><tr><td>Tryptone</td><td>Formedium</td><td>TRP03</td><td></td></tr><tr><td>Agar Bacteriological Grade</td><td>Applichem</td><td>A0949</td><td></td></tr><tr><td>Sf-900 II SFM medium</td><td>Gibco</td><td>10902-088</td><td></td></tr><tr><td>Grace&amp;rsquo;s Insect Medium, unsupplemented</td><td>Gibco</td><td>11595-030</td><td></td></tr><tr><td>Cellfectin II Reagent</td><td>Invitrogen</td><td>10362-100</td><td></td></tr><tr><td>MS medium including vitamins</td><td>Duchefa Biochemie</td><td>M0222</td><td></td></tr><tr><td>Sucrose</td><td>Duchefa Biochemie</td><td>S0809</td><td></td></tr><tr><td>Plant agar</td><td>Duchefa Biochemie</td><td>P1001</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Duchefa Biochemie</td><td>A0104</td><td>Toxic</td></tr><tr><td>Gentamycin sulphate</td><td>Duchefa Biochemie</td><td>G0124</td><td>Toxic</td></tr><tr><td>Ganciclovir</td><td>Invitrogen</td><td>I2562-023</td><td></td></tr><tr><td>Carbenicillin disodium</td><td>Duchefa Biochemie</td><td>C0109</td><td>Toxic</td></tr><tr><td>Kanamycin sulfate</td><td>Sigma</td><td>K4000</td><td>Toxic</td></tr><tr><td>Rifampicin</td><td>Duchefa Biochemie</td><td>R0146</td><td>Toxic &amp;ndash; 25 mg/ml stock in DMSO</td></tr><tr><td>Streptomycin sulfate</td><td>Duchefa Biochemie</td><td>S0148</td><td>Toxic</td></tr><tr><td>Spectinomycin dihydrochloride</td><td>Duchefa Biochemie</td><td>S0188</td><td></td></tr><tr><td>IPTG (isopropil-&amp;beta;-D-1-tiogalattopiranoside)</td><td>Sigma</td><td>I5502</td><td>Toxic</td></tr><tr><td>MES hydrate</td><td>Sigma</td><td>M8250</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biochemical</td><td>436994U</td><td></td></tr><tr><td>Acetosyringone</td><td>Sigma</td><td>D134406</td><td>Toxic &amp;ndash; 0.1 M stock in DMSO</td></tr><tr><td>Syringe (1 ml)</td><td>Terumo</td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fluka</td><td>63136</td><td></td></tr><tr><td>BAP (6-Benzylaminopurine)</td><td>Sigma</td><td>B3408</td><td>Toxic</td></tr><tr><td>NAA (Naphtalene acetic acid)</td><td>Duchefa Biochemie</td><td>N0903</td><td>Irritant</td></tr><tr><td>Cefotaxime</td><td>Mylan Generics</td><td></td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td>Adjust pH with 1 N HCl to make Tris-HCl buffer</td></tr><tr><td>HCl</td><td>Sigma</td><td>H1758</td><td>Corrosive</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014</td><td>1 M stock</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>PMSF (Phenylmethanesulfonylfluoride)</td><td>Sigma</td><td>P7626</td><td>Corrosive, toxic</td></tr><tr><td>Urea</td><td>Sigma</td><td>U5378</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td>Toxic</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P5927</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>DTT (Dithiothreitol)</td><td>Sigma</td><td>D0632</td><td>Toxic &amp;ndash; 1 M stock, store at -20 &amp;deg;C</td></tr><tr><td>CHAPS</td><td>Duchefa Biochemie</td><td>C1374</td><td>Toxic</td></tr><tr><td>Plant protease inhibitor cocktail</td><td>Sigma</td><td>P9599</td><td>Do not freeze/thaw too many times</td></tr><tr><td>SDS (Sodium dodecyl sulphate)</td><td>Sigma</td><td>L3771</td><td>Flammable, toxic, corrosive &amp;ndash; 10% stock</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Brilliant Blue R-250</td><td>Sigma</td><td>B7920</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>24137</td><td>Flammable</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>27221</td><td>Corrosive</td></tr><tr><td>Anti-Glutamic acid decarboxylase 65/67</td><td>Sigma</td><td>G5163</td><td>Do not freeze/thaw too many times</td></tr><tr><td>Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td><td>Sigma</td><td>A6154</td><td>Do not freeze/thaw too many times</td></tr><tr><td>Sf9 Cells</td><td>Life Technologies</td><td>11496</td><td></td></tr><tr><td>BL21 Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2530H</td><td></td></tr><tr><td>Protein A Sepharose</td><td>Sigma</td><td>P2545</td><td></td></tr><tr><td>Cell culture plates</td><td>Sigma</td><td>CLS3516</td><td></td></tr><tr><td>Radio Immuno Assay kit</td><td>Techno Genetics</td><td>12650805</td><td>Radioactive material</td></tr></tbody></table>

a comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems

植物为基础的系统被认为是用于生产重组蛋白作为其证据充分的电位为柔性，低成本生产高质量的，生物活性的产品的结果的有价值的平台。 在这项研究中，我们比较了在传统的发酵罐系的细胞培养物（细菌和昆虫）与基于植物的表达系统，无论是瞬时和稳定的靶人重组蛋白的表达。 每个平台，我们所描述的设置，优化和生产过程的长度，最终产品的质量和产量，我们评估了临时的生产成本，具体为所选靶重组蛋白质。 总的来说，我们的结果表明，细菌是不适合用于生产目标蛋白的，由于其不溶性包涵体内积累。另一方面，基于植物的系统是通用的平台吨帽允许生产所选蛋白质在较低的成本比杆状病毒/昆虫细胞系统。尤其是，稳定的转基因系显示最高产量的终产物和瞬时表达的植物最快流程开发。然而，并非所有的重组蛋白可受益于基于植物的系统中，但最好的生产平台应凭经验用逐案的方式来确定，如这里所述。

实验设计为靶重组蛋白在不同的生产系统中的异源表达在这里被描述。第一焦点是通过建立最佳条件对靶蛋白的每个系统中表达所述的建立的不同的平台。 靶蛋白，hGAD65mut的表达，诱导式三份大肠杆菌大肠杆菌文化。下列3小时的表达，在37℃，细菌细胞通过离心收集并通过超声处理裂解。离心步骤后，可溶性蛋白从不溶包涵体分离和初步分析表明，hGAD65mut在不溶性包?...

Watch this Scientific Journal Video about A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems at JoVE.com

不同生物工厂：细菌、昆虫细胞和植物系统中重组蛋白表达的比较分析生物学 |视频

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

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 ...

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23.4K 次观看.  University of Verona, Verona, Italy. 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.

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

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

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

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

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

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 ...

科研

遗传学

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 ...

生物化学

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

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

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

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

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

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

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

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 ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

基于海拉细胞自由表达系统中的表达<em&gt;疟原虫</em&gt;棒状体蛋白

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

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.

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

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