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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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+Cell+Migration+on+Artificial+Extracellular+Matrix+Proteins+Containing+Full-Length+Fibronectin+Domains.">Collective Cell Migration on Artificial Extracellular Matrix Proteins Containing Full-Length Fibronectin Domains.</a> Advanced Materials. 22 (46), 5271-5275 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIOMATERIALS:+Where+We+Have+Been+and+Where+We+are+Going.">BIOMATERIALS: Where We Have Been and Where We are Going.</a> Annual Review of Biomedical Engineering. 6 (1), 41-75 (2004).</li><li>Cai, L., Heilshorn, S. 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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</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>1M 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&#8482; 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

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

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, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

细胞外基质蛋白纤维的布里渊谱的制备

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...