Name: design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering
Uploaded: 2015-06-11T13:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

作者想感谢来自教育部ACRF一级（RG41）资金和南洋理工大学启动补助。低，Tjin由研究学生奖学金（RSS）南洋理工大学，新加坡资。

1.克隆重组质粒编码AECM蛋白质<ol><li>设计的功能域（ 例如 ，细胞结合结构域和弹性蛋白样重复序列）的氨基酸序列。设计限制位点侧翼的功能结构域的末端，以促进亚克隆使用自由软件根据软件指令（ 例如 ，http://biologylabs.utah.edu/jorgensen/wayned/ape/）。这里，选择唯一的限制性位点不存在于功能结构域来限制消化到预期位点。选择出现在多克隆位点的宿主载体的（MCS）的限制位点（ 例如 ，pET22b（+））用于亚克隆。 </li><li>反向翻译的氨基酸序列的成根据软件指令使用的免费访问的核苷酸序列。 （ 例如 ，http://www.bioinformatics.org/sms2/rev_trans.html）。确保密码子的优化E.大肠杆菌宿主。 </li><li>感兴趣的基因可以是珀切斯编市售为单链寡核苷酸（ 例如，如果寡核苷酸&lt;100个碱基对（bp））并进行DNA的退火（参见步骤1.4），以降低成本。否则，对于基因比100 bp的大，他们可以通过商业公司购买。 </li><li>寡核苷酸的DNA的退火<ol><li>退火的DNA寡聚体以获得所需的基因序列。溶解在DNA寡聚物缓冲液（10mM的Tris:2-氨基-2-（羟甲基） - 丙-1,3-二醇，pH为8.0，过滤）的DNA寡核苷酸至1微克/微升的最终浓度。 </li><li>添加4μl的每个低聚物的32微升的DNA退火缓冲液（10mM的Tris，100mM的NaCl和100纳米的MgCl 2），以获得总的40微升混合物。 </li><li>煮沸的水使用加热板的烧杯中并进行5分钟，95℃下浸入该混合物。取出烧杯并逐渐冷却整个设立一个泡沫塑料箱O / N。低聚物已退火并准备消化。 </li></ol></LI&gt; <li>添加相应的限制性内切酶消化退火的寡聚物（ 即 ，被称为插入物）和宿主载体（ 即 ，pET22b（+））分别。使用下面的配方（1-2微克的DNA，2微升各限制酶，5微升的10×限制性酶缓冲液中，加水至总50μl的混合物）为3-4小时，在37℃。 注:pET22b（+）质粒载体是氨苄青霉素抗生素耐药性，并且包含在C-末端具有6×组氨酸标记。的6×组氨酸标记存在于pET22b（+），使我们能够使用His标记的抗体通过免疫印迹以确定在步骤7中的靶蛋白。 </li><li>添加6X样染料给每个消化混合物。运行消化混合物分别包括在含有紫外荧光DNA染色1小时，在100伏可视化用UV光照射的1.2％琼脂糖凝胶的1.2％琼脂糖凝胶上的DNA梯。 </li><li>切片凝胶提取使用商业凝胶纯化试剂盒消化的DNA的产品。 ELUTe。使用最小体积的柱，以实现50-100纳克/微升最低DNA浓度。 </li><li>通过结扎消化的DNA插入物结合顺序的兴趣的基因（ 即，通过DNA退火所得弹性重复或细胞结合结构域）插入用T4连接酶用下列配方的质粒载体:（2微升载体，1微升T4连接酶的， 1.5微升T4连接缓冲液中，x微升刀片，10.5-X微升水，共15微升的混合物）。在室温下孵育连接混合物2小时。 注:矢量插入的摩尔浓度应改变以优化连接效率。在配方中描述为x刀片的体积取决于洗脱的DNA浓度对从步骤1.7。 </li><li> E.解冻大肠杆菌 DH5α的化学感受态细胞（或任何克隆株）在冰上。温暖的2xYT琼脂平板（ 表1）含氨苄青霉素（25微克/毫升）至37℃。 </li><li>变换使用细胞热休克: <ol><li>等份加入50μl感受态细胞转化为清洁，预冷的微量离心管中。吸管5微升（介于100 10pg至100毫微克）的连接混合物进入细胞，吹打轻轻上下混合。置于冰上的混合物20分钟。 </li><li>在42℃水浴中浸入含有细胞混合物的微离心管中进行2分钟，并返回到在冰上2分钟。时间的浸渍持续时间，以减少热量的损害细胞。 </li><li>加入500微升SOC培养基（ 表1）的进入微离心管中并在37℃振荡1小时。 </li><li>蔓延50将500μl细胞/连接混合物到的2xYT琼脂板，其已经预先温热至RT，含有氨苄青霉素（25微克/毫升），并孵育板在37℃CO倒置/ N（12-16小时） 。 </li></ol></li><li>第二天，挑DNA克隆使用干净的枪头琼脂平板上。生长的菌落在5毫升2YT培养基（ 表1）含氨苄青霉素（25微克/毫升）O / N在37℃的CO / N（12-16小时）振荡（225转）。 </li><li>次日，使用质粒分离试剂盒提取DNA质粒用于根据制造商的协议拾取每一集落。洗脱用50μl水的DNA。 </li><li>执行与使用下列配方限制酶测试消化:（5微升的DNA，0.2微升每个限制酶，1微升10×限制性酶缓冲液和补足水，共10微升混合物），孵育2小时，在37℃下屏幕为菌落可能含有对1.2％琼脂糖凝胶插入并运行如步骤1.6。 注:在消化，一个成功的结扎应导致两个频带，分别用于载体和插入其对应于它们各自的分子量（ 图1）。 </li><li>使用T7启动子向前发送可能菌落用于DNA测序和反向引物序列发生器商用。 </li></ol><ol><li>从测序结果，选择一个菌落已成功连接，并使用该DNA的质粒转化入大肠杆菌大肠杆菌表达宿主。 </li><li> E.解冻大肠杆菌表达菌株（BL21（DE3）pLysS中）在冰上。同时，含有氨苄青霉素（25微克/ ml）和氯霉素（34微克/毫升）至RT温暖琼脂平板上。 注:pLysS中的质粒存在于细菌菌株BL21（DE3）pLysS中含有氯霉素抗性基因。所述pLysS中质粒含有T7阻遏物基因被组成型表达，以限制AECM蛋白的渗漏表达。氯霉素有必要选择为细菌细胞，它包含pLysS中培养期间。 </li><li>重复步骤1.10到获得转化E.大肠杆菌细胞的准备表达人工蛋白质。包裹琼脂平板在封口膜，并存储在4倒置 6℃持续长达一个月。 </li></ol>蛋白质AECM 3.细菌表达<ol><li>参考图2，选择从转化的琼脂板上的菌落，用枪头，接种到10ml无菌了不起肉汤（TB）培养基含有在试管既氨苄青霉素和氯霉素抗生素（ 表1）。孵育在37℃的CO / N（12-16小时）此起始培养振荡下225转。 </li><li>将10毫升起子培养的成补充有在一个3升的Erlenmeyer烧瓶中的相同抗生素1升新鲜无菌TB介质。孵育培养在37℃下与在225 rpm振摇2-3小时并观察培养物（OD 600）的光密度吹打1毫升培养到空反应杯进行读取达到0.6-0.8。节省1 ml培养之前进行诱导SDS-PAGE表征。 <ol><li>为了测量细胞培养物的OD 600，准备1小瓶TB介质在比色杯一个空白的测量。另外，转移1毫升从培养瓶中培养成一个新的空试管。测量使用针对空白对照分光光度计培养物的光密度在600nm的吸光度。 </li><li>通过将1ml的培养样品放入1.5ml微量离心管中，离心12,000×g离心2分钟，保存的样品（用于随后的SDS-PAGE分析），并倾析上清液。 </li></ol></li><li>诱导用异丙β-D-1-硫代半乳糖苷（IPTG）培养至1mM的终浓度，并在37℃下在225 rpm振摇另外4小时。节省1 ml培养的诱导月底在4小时的SDS-PAGE表征。 </li><li>通过在12,000×g离心在4℃下将所述培养至1L离心瓶，离心30分钟收获细胞。弃去上清液，称量细胞沉淀，重悬在TEN缓冲液（1米的Tris，0.01M EDTA，0.1 M氯化钠，pH值= 8.0），以0.5克/毫升。 </li></ol> 4.裂解细菌培养<ol><li>冻结再悬浮培养细胞在-80°CO / N。解冻在水浴冷冻细胞培养物在室温或在冰上裂解细胞。加10微克/毫升脱氧核糖核酸酶I（DNA酶I），10微克/毫升核糖核酸酶A（RNA酶I）中，和50微克/毫升苯甲磺酰氟（PMSF），而解冻并均化缓慢搅拌下的溶液中。 </li><li>毕竟再悬浮细胞被解冻，调节溶液至pH 9.0，以增加在水中12中的蛋白质的溶解度。添加6N的氢氧化钠滴加在搅拌下在冰上以实现均匀的一致性。裂解通过使用2毫米直径的平末端，5秒脉冲在冰上20分钟超声波破碎。 </li><li>离心在12000×g离心细胞溶液进行30分钟，在4℃。将上清液转移到一个干净的空瓶子，并储存于4℃下纯化后。 </li><li>同时，再次重悬细胞沉淀用TEN缓冲液，并重新冷冻在-80℃。至完整细胞裂解，重复冷冻/解冻和超声处理过程高达三倍。保存20微升细胞裂解液进行SDS-PAGE表征。 </li></ol> 5. AECM纯化的蛋白质的逆转变自行车<ol><li>整理从步骤4.3细胞裂解物，并继续以纯化使用ITC，类似于用于弹性基蛋白21的纯化AECM蛋白质。周期将与不同温度，这是4℃下进行（以下称为"冷"）和37℃下分别（称为"暖"）周期。 </li><li>分裂细胞裂解物于50毫升离心瓶，并离心以40,000×g离心2小时，在4℃。细胞沉淀的外表应该为深褐色，看起来松软。 </li><li>通过移液除去上清液得到一个干净的分离沉淀。收集在清洁离心瓶中的上清液，加入氯化钠使之达到1M的终浓度（最大为3米）。温暖该溶液至37℃2小时振荡下225转。 注:加入的NaCl将触发AECM引起聚集的弹性成分的过渡。上清液会变成浑浊。如果蛋白质浓度足够高时，白色泡沫状蛋白可以在离心瓶的侧面可以看到。 </li><li>离心从步骤5.3上清液以40,000×g离心2小时，在37℃。倒出上清液。粉碎，使用金属刮刀将沉淀，并使用磁力搅拌棒和板重悬在冰冷的灭菌蒸馏水的沉淀位（50毫克/毫升）在剧烈搅拌下O / N在4℃。粒料应完全溶解。 </li><li>重复步骤5.2至5.4为3到5次，得到该蛋白质的纯度更高。 </li><li>纯化的最后一个循环后，通过透析它对蒸馏水在4℃脱盐的蛋白质溶液。透析针对水的蛋白质溶液2-3天的变化在水中，每4小时或8小时的O / N。保存20微升纯化的蛋白SDS-PAGE，并在-80°C，直到进一步利用冻干纯化的蛋白质和商店的休息。 </li></ol> AECM蛋白6.表征使用的SDS-PAGE电泳<ol><li>制备在12％SDS-PAGE凝胶（如果分子量大，则使用8％SDS-PAGE凝胶进行更好的分离）。加2×SDS上样缓冲液到每个样品中，加热样品10分钟，在100℃下并运行样品在凝胶上进行1小时，在100伏或直至相应于AECM蛋白质的分子量的蛋白质梯到达的中间凝胶。 </li><li>检索来自SDS-PAGE设置凝胶和与卷浸没凝胶1小时在培养皿中添加考马斯亮蓝染色（ 表2）。漂洗凝胶在脱色溶液（ 表2）5分钟上的摇杆。更改脱色液，并继续脱色凝胶与摇摆，直到T的背景他凝胶变得清晰。蛋白质条带应清晰可见。 </li><li>比较所述目标蛋白的位置针对该蛋白质梯以确定目标蛋白质的分子量。 </li><li>为了进一步证实了靶蛋白的存在，进行免疫印迹如在步骤7。 </li></ol> AECM蛋白7.使用表征免疫印迹<ol><li>运行在12％SDS-PAGE凝胶样品按与没有染色部分6。 </li><li>切断硝基纤维素膜的尺寸比SDS-PAGE凝胶略大。切4张该滤纸为相同大小。 </li><li>湿一块与西方转移缓冲液的滤纸（20％体积/体积甲醇，25毫摩尔Tris，190 mM的甘氨酸，pH值8.3），放置在滤纸的顶部的SDS-PAGE凝胶，接着另一片滤纸的话，硝酸纤维素膜和最终其他两片滤纸。保证了整个设置被淹没足够的西部转移的缓冲。 注:凝胶和膜不应该在此时的时间接触蛋白质可以结合在接触时的膜。 </li><li>转移的蛋白到硝酸纤维素膜通过将两个滤纸成西部半干转移单元，其次是硝酸纤维素膜，并小心地将内和在膜的SDS-PAGE凝胶，最后在放置最后两个湿滤纸在SDS-PAGE凝胶。运行在45毫安，30分钟的西部转移。加入缓冲液，如果电压高于30 V. 注:优选放置在膜的SDS-PAGE凝胶与一种尝试，并避免不必要地移动的凝胶在膜蛋白质可以在接触时结合到膜上。 </li><li>检索和阻断硝酸纤维素膜用2小时在RT封闭液（5％脱脂乳的PBS pH为7.4，用滤纸过滤）。处置封闭缓冲液，并添加PBS冲洗。 注:更改为新的手套，以避免污染与不需要prote膜插件。 </li><li>初级孵育反他与PBS稀释抗体在1:1000在RT 1小时与摇摆。在一个体积可能淹没整个膜，例如，用稀释比为1制备混合物:1000，加入1微升抗体（母液浓度:1毫克/毫升），以999微升PBS中1毫升总混合物。 </li><li>冲洗用5毫升的PBST（PBS中含有0.1％吐温20）的膜。 </li><li>孵育缀合至辣根过氧化物酶（HRP）与稀释于PBS 1二级抗体:5000在RT 1小时与摇摆。在一个体积可能淹没整个膜，例如，用稀释比为1制备混合物:5000，加入1微升抗体（母液浓度:1毫克/毫升）至4999微升PBS中5ml的总混合物。 </li><li>用5毫升的PBST洗膜，重复两次。 </li><li>适用的化学发光底物与膜带卷以覆盖膜，并按照所用的基片的说明孵化。 注:蛋白质检测基板的灵敏度可能会发生变化，我们在事件建议具有最大灵敏度的基板对于非常低的信号增加抗体浓度不增加信号。 </li><li>拍摄使用的是CCD相机成像基础化学发光信号。调节初级和次级抗体稀释比如果信号弱和非特异性。在封闭液，如果背景信号为高电平孵育更长。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+ECM-mimetic+materials+using+protein+engineering.">Designing ECM-mimetic materials using protein engineering.</a> Acta Biomater. 10 (4), 1751-1760 (2014).</li><li>Annabi, N., 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=Elastomeric+recombinant+protein-based+biomaterials.">Elastomeric recombinant protein-based biomaterials.</a> Biochemical Engineering Journal. 77, 110-118 (2013).</li><li>Heilshorn, S. C., DiZio, K. A., Welsh, E. R., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+adhesion+to+the+fibronectin+CS5+domain+in+artificial+extracellular+matrix+proteins.">Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins.</a> Biomaterials. 24 (23), 4245-4252 (2003).</li><li>Simnick, A. J., Lim, D. W., Chow, D., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+and+Biotechnological+Applications+of+Elastin-Like+Polypeptides.">Biomedical and Biotechnological Applications of Elastin-Like Polypeptides.</a> Polymer Reviews. 47 (1), 121-154 (2007).</li><li>Tjin, M. S., Chua, A. W. C., Ma, D. R., Lee, S. T., Fong, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Epidermal+Keratinocyte+Cell+Response+on+Integrin-Specific+Artificial+Extracellular+Matrix+Proteins.">Human Epidermal Keratinocyte Cell Response on Integrin-Specific Artificial Extracellular Matrix Proteins.</a> Macromolecular Bioscience. 14 (8), 1125-1134 (2014).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+opportunities+for+tissue-engineered+skin.">Progress and opportunities for tissue-engineered skin.</a> Nature. 445 (7130), 874-880 (2007).</li><li>Kariya, Y., 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=Differential+regulation+of+cellular+adhesion+and+migration+by+recombinant+laminin-5+forms+with+partial+deletion+or+mutation+within+the+G3+domain+of+&#945;3+chain.">Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of &#945;3 chain.</a> J Cell Biochem. 88 (3), 506-520 (2003).</li><li>Shang, M., Koshikawa, N., Schenk, S., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LG3+module+of+laminin-5+harbors+a+binding+site+for+integrin+&#945;3&#914;1+that+promotes+cell+adhesion,+spreading,+and+migration.">The LG3 module of laminin-5 harbors a binding site for integrin &#945;3&#914;1 that promotes cell adhesion, spreading, and migration.</a> J Biol Chem. 276 (35), 33045-33053 (2001).</li><li>Hassouneh, W., Christensen, T., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-like+polypeptides+as+a+purification+tag+for+recombinant+proteins.">Elastin-like polypeptides as a purification tag for recombinant proteins.</a> Current Protocols in Protein Science. , 6.11.1-6.11.16 (2010).</li><li>Keeley, F., Mecham, R., Keeley, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+4.">Ch. 4.</a> Evolution of Extracellular Matrix.Biology of Extracellular Matrix. , 73-119 (2013).</li><li>Le, D. H. T., 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=Self-Assembly+of+Elastin-Mimetic+Double+Hydrophobic+Polypeptides.">Self-Assembly of Elastin-Mimetic Double Hydrophobic Polypeptides.</a> Biomacromolecules. 14 (4), 1028-1034 (2013).</li><li>MacEwan, S. R., Hassouneh, W., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-chromatographic+Purification+of+Recombinant+Elastin-like+Polypeptides+and+their+Fusions+with+Peptides+and+Proteins+from+Escherichia+coli.">Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli.</a> Journal of visualized experiments: JoVE. (88), e51583 (2014).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li></ol>

作者宣称，他们有没有竞争的财务权益。

重组蛋白质工程是一种通用的技术来创建使用自下而上的方法新的蛋白质材料。的基于蛋白质的材料可根据感兴趣的应用被设计成具有多种功​​能，量身定做。由于增加的进步中克隆和表达的技术，它已成为比较简单（和成本有效），以在一个可再现的和可扩展的方式创建各种人造蛋白质。弹性蛋白样结构域已在许多人造蛋白质被结合，以用作纯化标记，以及赋予机械性能。包含弹性蛋白样序?...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table><tbody><tr><td>pET22b (+)</td><td>Novagen</td><td>69744</td><td>T7 expression vectors with resistance to ampicillin&amp;nbsp;</td></tr><tr><td>BL21(DE3)pLysS&amp;nbsp;</td><td>Invitrogen</td><td>C6060-03</td><td>additional antibiotics - chloramphenicol</td></tr><tr><td>Isopropyl-beta-D-thiogalactoside (IPTG)</td><td>Gold Biotechnology</td><td>I2481C</td><td>1 M stock solution with autoclaved water, make fresh prior to induction.</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>plasmid isolation kit</td></tr><tr><td>T4 ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>Ampicillin</td><td>Affymetrix</td><td>11259</td><td></td></tr><tr><td>Chloramphenicol</td><td>Affymetrix</td><td>23660</td><td></td></tr><tr><td>Zymoclean&amp;trade; gel DNA recovery kit</td><td>Zymo Research</td><td>D4001</td><td></td></tr><tr><td>XL10-gold strain</td><td>Agilent Technologies</td><td>200315</td><td></td></tr></tbody></table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

在设计包含弹性蛋白样重复融合蛋白，它维持一个整体弹性内容中，融合蛋白18的足够大的部分是很重要的。这是为了确保该融合蛋白构建体保留其弹性蛋白样特征，以便使用ITC进行纯化。的AECM蛋白质设计和本节中所述的序列被明确取自工作由Tjin 等人14。在这项工作中，三AECM蛋白被成功地克隆到pET22b（+）表达载体。连续结扎首先开始与结扎弹力重复插入到pET载体，随后通...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

design-construction-artificial-extracellular-matrix-aecm-proteins

Research

JoVE Journal

Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

8.8K 次观看.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

视频: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

科研

生物工程

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

发育生物学

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

自组装血管化人体皮肤等价物的生成

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

<meta charset="utf8"></head><body>高效的 3D 皮肤模型，用于灵活的研究应用

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

<meta charset="utf8"></head><body>作者聚焦：使用经济高效的 3D 细胞模型增强皮肤模型的多样性

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

设计和人工细胞外基质的构建（AECM）蛋白质

摘要

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此视频中的章节

组织工程：建设多细胞三维支架的层状细胞片交货

一个完整的皮肤缺损模型，以评估生物材料的血管化

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

工程三维细胞化胶原凝胶的血管组织再生

提高2D和3D皮肤

工程三维上皮组织细胞外基质中嵌入

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

牺牲纤维膜细胞培养生产细胞外基质纤维

自组装血管化人体皮肤等价物的生成

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养

设计和人工细胞外基质的构建（AECM）蛋白质

摘要

探索更多视频

此视频中的章节

组织工程：建设多细胞三维支架的层状细胞片交货

一个完整的皮肤缺损模型，以评估生物材料的血管化

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

工程三维细胞化胶原凝胶的血管组织再生

提高2D和3D皮肤

工程三维上皮组织细胞外基质中嵌入

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

牺牲纤维膜细胞培养生产细胞外基质纤维

自组装血管化人体皮肤等价物的生成

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养