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>

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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?...

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 border="0" cellpadding="0" cellspacing="0" width="667">
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			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">24137</td>
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			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">27221</td>
			<td dir="LTR" style="width:223px;">Corrosive</td>
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		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:191px;">Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">A6154</td>
			<td dir="LTR" style="width:223px;">Do not freeze/thaw too many times</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:191px;">Sf9 Cells</td>
			<td dir="LTR" style="width:111px;">Life Technologies</td>
			<td dir="LTR" style="width:143px;">11496</td>
			<td dir="LTR" style="width:223px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:191px;">BL21 Competent E.coli</td>
			<td dir="LTR" style="width:111px;">New England Biolabs</td>
			<td dir="LTR" style="width:143px;">C2530H</td>
			<td dir="LTR" style="width:223px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Protein A Sepharose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:143px;">P2545</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Cell culture plates&#160;</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:143px;">CLS3516</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Radio Immuno Assay kit</td>
			<td>Techno Genetics</td>
			<td>12650805</td>
			<td style="width:223px;">Radioactive material&#160;</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

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

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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23.2K 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

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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 ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...