这项工作是由美国国家科学基金会的研究三角MRSEC（DMR-1121107）的支持。

1，ELP表达<ol><li>接种3毫升无菌极品肉汤媒体与DMSO股票或大肠杆菌的琼脂平板上菌落大肠杆菌适合包含下的T7-lac启动子的控制所需的ELP-质粒编码蛋 ​​白的表达。添加适当的抗生素和连续200rpm振荡孵育37℃过夜（O / N）。 </li><li>加入1 ml的O / N培养1升的4-L烧瓶极品肉汤的无菌介质中。添加适当的抗生素和孵化，在37℃下24小时连续200rpm振荡。注：看到与T7-lac启动子的“漏”基线表达足以让许多电子学习产品的高水平表达，如果这样的文化培养24小时。然而，更传统的方法都可以使用，其特征在于：蛋白质的表达进一步通过加入0.2-1 1mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导，当培养物的光密度达到0.6。 </li&gt; <li>从4-L烧瓶中转移到培养1升的离心瓶中并离心机在4℃下进行15分钟，在2000×g下。 </li><li>弃上清。注：如果超过1升培养物生长，他们可以通过添加另一个升培养物的沉淀，并重复步骤1.3中冷凝。高达2升培养物可以被浓缩成单个细胞沉淀。 </li><li>悬浮于磷酸盐缓冲盐水（PBS），水，或其它所需的缓冲颗粒达到45 ml的细胞悬浮液，并转移到50毫升锥形管中。 </li></ol> 2 ELP净化：预备步骤和第一轮逆转换的自行车<ol><li>超声处理的悬浮细胞沉淀，总共9分钟的“上”10秒和20秒“关断”时的85瓦特的输出功率的周期，同时保持样品在冰上。简单地说让样品冷却冰，然后重复9分钟循环一次。注意：如果ELP预计将有一个非常低的T T </子&gt;“关断”的时间间隔可以增加至40秒，以避免上述的T T溶液的加热。 </li><li>裂解液转移至50毫升圆底离心管中。 </li><li>加入2毫升10％（W / V）聚乙烯亚胺（PEI）为每公升文化，并摇匀。注意：PEI是带正电荷的聚合物，其有助于在带负电荷的遗传污染物的缩合。不要执行此步骤如果ELP的负电荷，由于PEI还可以凝结，取出ELP产品。 </li><li>离心机在16,000 xg 4℃，10分钟。 </li><li>将上清转移至50 mL的干净圆底离心管中，弃沉淀。 </li><li>可选的“烘烤”的步骤：孵育样品在60°C下10分钟以变性蛋白污染物，然后转移到4℃或冰上，直到ELP完全resolubilized。离心机的样品在以16,000×g下4℃放置10分钟。将上清转移到CLEAN50毫升圆底离心管中，弃去沉淀。注意：如果肽或蛋白融合到电子学习产品，并有望在高架热条件变性不要执行此步骤。这可能会导致对ELP过渡成为热不可逆的或可能破坏稠合基团的活性。 </li><li>诱导与另外结晶氯化钠（不超过3公尺）的ELP过渡。可替换地，柠檬酸钠（不超过0.3 M），可用于具有较高的T T S或低收率电子学习。注意： 该解决方案应该把混浊一旦盐溶解，说明ELP从溶液相分离。具有高T T的甚亲水电子学习产品可能需要一定程度的加热，与加成盐的组合，诱导对ELP的相分离在ELP过渡的这一初步的诱导。 </li><li>离心机在室温temperatue（RT）下以16,000×g离心（热自旋）10分钟。注意：一个ELP颗粒B应该要Ë观察，其大小取决于ELP的表达产量。这ELP颗粒可能会在第一次出现不透明的，但冷却后会出现半透明的，可能是棕色的。对ELP颗粒将变得更加无色的净化所得款项及污染物去除。 </li><li>弃去上清液，加入1-5毫升所需的缓冲区来沉淀。通过移液重悬沉淀或将管在4℃下旋转 注意：缓冲器的所需体积将取决于ELP粒料的大小，从而ELP的表达，可在其中添加缓冲足够量以允许所述ELP粒料再增的产率。电子学习与纯化水中的10mM的Tris（2 - 羧乙基）膦盐酸盐（TCEP-HCl中）在pH为7，以消除污染的蛋白质二硫键相互作用高半胱氨酸含量的好处。电子学习产品与净化的pH值调节高电荷含量的利益，以中和其电荷和减少元素ctrostatic互动与杂蛋白。 </li><li>转移1毫升分装再悬浮的ELP溶液倒入洁净的1.5 ml离心管中。 </li><li>离心机在4℃下在16,000 XG（冷旋）10分钟。小颗粒不溶性杂质应得到遵守。 </li><li>将上清转移到一个干净的试管，弃去沉淀。 </li></ol> 3，ELP净化：反后过渡骑自行车的回合<ol><li>诱导用热和/或添加NaCl对ELP过渡。样品可以培养在热块或在水浴中15分钟，在上面的T T的温度。然而，如果对ELP融合到蛋白质（排除使用热），或者如果ELP具有高T T，不能用热单独达到的NaCl 5M的溶液可以逐滴加入以诱导ELP过渡。注：该解决方案应该转多云，表明ELP从溶液相分离。 </li><li&gt;离心机在16,000 XG（热旋）10分钟。如果热量在步骤3.1中使用，上面的这是需要引起对ELP转变的温度下进行这种离心步骤。如果单独的NaCl在步骤3.1中使用，在室温下执行该离心步骤。注：ELP颗粒应得到遵守。 </li><li>弃去上清液，加入500-750微升所需的缓冲液中，并通过移液重悬沉淀或将管在4℃下旋转</li><li>离心机在4℃下在16,000 XG（冷旋）10分钟。小颗粒不溶性杂质应得到遵守。 </li><li>将上清转移到一个干净的试管，弃去沉淀。 </li><li>直到没有杂质沉淀在步骤3.4观察重复步骤3.1至3.5。 </li></ol> 4，后期净化处理（可选） <ol><li>如果需要的话，透析样品对一个合适的缓冲液，以从纯化ELP溶液除去过量的盐。注意：电子学习产品被冷冻干燥应透析双蒸二 OO / N在4°C。 </li><li>如果需要，可通过冷冻干燥的O / N。冷冻前的溶液在-80℃下30分钟的冷冻干燥ELP用于长期存储注意：冻干不应被用于电子学习与所附的蛋白质。 </li><li>如果需要，可通过除去ELP标签回收游离肽或蛋白由肽或蛋白质的ELP融合： <ol><li>消化的肽或蛋白质的ELP融合与生物素化的蛋白酶（所推荐的蛋白酶供应商）或与相对应的蛋白酶位点的肽或蛋白与ELP之间的基因而设计的蛋白酶ELP融合。 </li><li>如果适用的话，用链亲和素修饰的磁珠（所推荐的蛋白酶供应商）除去生物素化的蛋白酶。 </li><li>诱导裂解ELP和/或蛋白酶ELP融合了逐滴加入的5M NaCl溶液的过渡。 </li><li>离心机在室温下以16,000×g的10分钟。注：ELP颗粒应得到遵守。 </li><li>收集上清液并弃去沉淀ELP。透析的上清液对所期望的缓冲液，或者使用离心过滤器，以便进行缓冲液交换，从纯肽或蛋白质溶液中除去高浓度的盐。 </li><li>确认来自ELP标签通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）游离肽或蛋白质的完全切割。 </li></ol></li></ol> 5特性：SDS-PAGE <ol><li>通过混合10微升ELP的稀释在水中用10微升含2 - 巯基乙醇的样品缓冲液中制备的ELP样品进行SDS-PAGE。注：40微克ELP的往往是足够的，以评估对ELP产品的大小和纯度。 </li><li>孵育样品在100℃下持续2分钟，然后加载到聚丙烯酰胺凝胶连同一个适当大小的蛋白质的阶梯。 </li><li>运行该凝胶在180 V开始，染料接近凝胶（约45分钟）的底部。 </li><li>染色凝胶用5分钟0.5M的氯化铜溶液，同时摇动。注意： 电子学习产品通常不被考马斯亮蓝染色显现，一些ELP序列不具有这种染料相互作用。考马斯亮蓝主要交互的精氨酸残基和弱酸和其它碱性氨基酸残基，组氨酸和赖氨酸-和芳族残基，色氨酸，酪氨酸和苯丙氨酸26交互。因而电子学习和肽或蛋白ELP融合物可染色用考马斯亮蓝仅当它们的主序列包括这些特定残基的足够数量。 </li><li>验证ELP带的分子量对应于从ELP基因，因而没有多余的杂质带都存在预期的理论分子量。注：某些电子学习产品迁移具有明显分子量比他们预期的9兆瓦，27高者可达20％。 </li></ol> 6性质：基质辅助激光解吸/电离飞行时间质谱的 - 飞行时间质谱（MALDI-TOF-MS） <ol><li>制备ELP溶液中含有0.1％三氟乙酸来实现的约25微米的ELP浓度为50％乙腈水溶液。注意：此解决方案应该是免费的盐，所以必须ELP纯化后进行透析，H 2 O </li><li>制备饱和芥子酸中含有0.1％三氟乙酸的50％乙腈水溶液中的基质溶液。 </li><li>加入1μl的ELP溶液到9微升的基质溶液，以达到约2.5皮摩尔/微升的ELP浓度。注：此混合物可与基质溶液被进一步稀释，以优化MALDI-TOF信号。 </li><li>存款1-2微升ELP-矩阵混合物在金属MALDI板，让现场完全干燥。 </li><li>获得5-10谱与MALDI-TOF确定质荷对ELP的比（m / z）和推断其测量兆瓦。 </li></ol> 7。Characterization为程序升温比浊法和动态光散射<ol><li>确定ELP通过程序升温比浊法的T T： <ol><li>备中所需的缓冲ELP溶液的稀释系列（ 例如 ，从5-100微米）。 </li><li>使用温度控制的紫外可见分光光度计，测量光密度（OD）在350nm处以上以1℃/ min的升温速率的温度范围内（ 如 20-80℃）。注意：如果没有增加外径是看到了T T可能会超过仪器的温度上限。表观T T可以通过加入NaCl降低。从1开始，M氯化钠和直到ELP过渡是一个可测量的温度范围内检测到增加步长为0.5M的。 </li><li>通过测量降低外径比相同的温度范围内以1℃/ min的冷却速度验证ELP过渡的可逆性。 </li><li>确定同源的T T聚合物ELP通过识别浊度曲线的拐点（OD值作图温度）。注：此温度可以从OD曲线，其中对应于所述导数的最大值的温度被定义为T T的导数很容易地确定。更复杂的电子学习产品架构将显示更为复杂的浊度型材和7.2节所述，应进一步分析。 </li></ol></li><li>可显示更为复杂的热性能（ 例如 ，ELP二嵌段共聚物的自组装成球状胶束）电子学习产品的附加 ​​特性是通过动态光散射（DLS）来实现： <ol><li>制备ELP溶液中所需的缓冲区，并通过过滤与20-450 nm的孔径合格的试样。注：过滤器孔径的选择将依赖于有无，大小，和在溶液中的任何ELP的组件的稳定性。最小的可行的过滤孔径是优选的，以消除污染物，如尘土，使尘土减少DLS测量的质量。当时，通过更小的过滤器的孔径传递一个电子学习解决方案显著性经历了一个较大的过滤孔径应该被使用。 </li><li>测量的流体动力学半径（R H）以上，对应于在浊度更新确定感兴趣的区域中的温度范围内进行。注：ELP unimers通常有一个R H &lt;10纳米，纳米粒子装配有R H〜20-100纳米，聚集有一个R H&gt; 500纳米。在R H每次增加应该对应于在350nm处通过UV-Vis分光光度法作为温度的函数，它正比于颗粒的尺寸测量的增加OD。 </li></ol></li></ol>

<ol>
	<li>Urry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+Chemistry+of+Biological+Free+Energy+Transduction+As+Demonstrated+by+Elastic+Protein-Based+Polymers.">Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers.</a> J. Phys. Chem. B. 101, 11007-11028 (1997).</li><li>Sallach, R. E., Conticello, V. P., Chaikof, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+recombinant+elastin-like+protein+in+pichia+pastoris.">Expression of a recombinant elastin-like protein in pichia pastoris.</a> Biotechnology progress. 25, 1810-1818 (2009).</li><li>Schipperus, R., Teeuwen, R. L., Werten, M. W., Eggink, G., de Wolf, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+production+of+an+elastin-like+polypeptide+by+Pichia+pastoris.">Secreted production of an elastin-like polypeptide by Pichia pastoris.</a> Appl Microbiol Biotechnol. 85, 293-301 (2009).</li><li>Schipperus, R., Eggink, G., de Wolf, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+of+elastin-like+polypeptides+with+different+transition+temperatures+by+Pichia+pastoris.">Secretion of elastin-like polypeptides with different transition temperatures by Pichia pastoris.</a> Biotechnology progress. 28, 242-247 (2012).</li><li>Herzog, R. W., Singh, N. K., Urry, D. W., Daniell, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+synthetic+protein-based+polymer+(elastomer)+gene+in+Aspergillus+nidulans.">Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans.</a> Appl Microbiol Biotechnol. 47, 368-372 (1997).</li><li>Conley, A. J., Joensuu, J. J., Jevnikar, A. M., Menassa, R., Brandle, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+elastin-like+polypeptide+fusions+for+expression+and+purification+of+recombinant+proteins+in+plants.">Optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants.</a> Biotechnology and bioengineering. 103, 562-573 (2009).</li><li>Conrad, U., 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=ELPylated+anti-human+TNF+therapeutic+single-domain+antibodies+for+prevention+of+lethal+septic+shock.">ELPylated anti-human TNF therapeutic single-domain antibodies for prevention of lethal septic shock.</a> Plant biotechnology journal. 9, 22-31 (2011).</li><li>Kaldis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+production+of+human+interleukin-10+fusions+in+tobacco+cell+suspension+cultures.">High-level production of human interleukin-10 fusions in tobacco cell suspension cultures.</a> Plant biotechnology journal. 11, 535-545 (2013).</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, 357-367 (2002).</li><li>McDaniel, J. R., Mackay, J. A., Quiroz, F. G., 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=Recursive+directional+ligation+by+plasmid+reconstruction+allows+rapid+and+seamless+cloning+of+oligomeric+genes.">Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.</a> Biomacromolecules. 11, 944-952 (2010).</li><li>Amiram, M., Quiroz, F. G., Callahan, D. J., 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=A+highly+parallel+method+for+synthesizing+DNA+repeats+enables+the+discovery+of+'smart'+protein+polymers.">A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers.</a> Nat Mater. 10, 141-148 (2011).</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=Quantification+of+the+effects+of+chain+length+and+concentration+on+the+thermal+behavior+of+elastin-like+polypeptides.">Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides.</a> Biomacromolecules. 5, 846-851 (2004).</li><li>Urry, D. W., 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=Temperature+of+Polypeptide+Inverse+Temperature+Transition+Depends+on+Mean+Residue+Hydrophobicity.">Temperature of Polypeptide Inverse Temperature Transition Depends on Mean Residue Hydrophobicity.</a> Journal of the American Chemical Society. 113, 4346-4348 (1991).</li><li>Urry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+change+in+Gibbs+free+energy+for+hydrophobic+association+-+Derivation+and+evaluation+by+means+of+inverse+temperature+transitions.">The change in Gibbs free energy for hydrophobic association - Derivation and evaluation by means of inverse temperature transitions.</a> Chem Phys Lett. 399, 177-183 (2004).</li><li>Cho, 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=Effects+of+Hofmeister+anions+on+the+phase+transition+temperature+of+elastin-like+polypeptides.">Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides.</a> The journal of physical chemistry. B. 112, 13765-13771 (2008).</li><li>Kim, B., 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=Allosteric+actuation+of+inverse+phase+transition+of+a+stimulus-responsive+fusion+polypeptide+by+ligand+binding.">Allosteric actuation of inverse phase transition of a stimulus-responsive fusion polypeptide by ligand binding.</a> J Am Chem Soc. 130, 17867-17873 (2008).</li><li>Hassouneh, W., Nunalee, M. L., Shelton, M. C., 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=Calcium+binding+peptide+motifs+from+calmodulin+confer+divalent+ion+selectivity+to+elastin-like+polypeptides.">Calcium binding peptide motifs from calmodulin confer divalent ion selectivity to elastin-like polypeptides.</a> Biomacromolecules. 14, 2347-2353 (2013).</li><li>Nettles, D. L., Chilkoti, A., Setton, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+elastin-like+polypeptides+in+tissue+engineering.">Applications of elastin-like polypeptides in tissue engineering.</a> Adv Drug Deliv Rev. 62, 1479-1485 (2010).</li><li>MacEwan, S. R., 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:+biomedical+applications+of+tunable+biopolymers.">Elastin-like polypeptides: biomedical applications of tunable biopolymers.</a> Biopolymers. 94, 60-77 (2010).</li><li>McDaniel, J. R., Callahan, D. J., 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=Drug+delivery+to+solid+tumors+by+elastin-like+polypeptides.">Drug delivery to solid tumors by elastin-like polypeptides.</a> Adv Drug Deliv Rev. 62, 1456-1467 (2010).</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> Curr Protoc Protein Sci. , (2010).</li><li>Shamji, M. F., 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=Development+and+characterization+of+a+fusion+protein+between+thermally+responsive+elastin-like+polypeptide+and+interleukin-1+receptor+antagonist:+sustained+release+of+a+local+antiinflammatory+therapeutic.">Development and characterization of a fusion protein between thermally responsive elastin-like polypeptide and interleukin-1 receptor antagonist: sustained release of a local antiinflammatory therapeutic.</a> Arthritis Rheum. 56, 3650-3661 (2007).</li><li>Hassouneh, W., 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=Unexpected+multivalent+display+of+proteins+by+temperature+triggered+self-assembly+of+elastin-like+polypeptide+block+copolymers.">Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers.</a> Biomacromolecules. 13, 1598-1605 (2012).</li><li>Lan, D. M., 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=An+improved+nonchromatographic+method+for+the+purification+of+recombinant+proteins+using+elastin-like+polypeptide-tagged+proteases.">An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases.</a> Analytical Biochemistry. 415, 200-202 (2011).</li><li>Bellucci, J. J., Amiram, M., Bhattacharyya, J., McCafferty, 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=Three-in-one+chromatography-free+purification,+tag+removal,+and+site-specific+modification+of+recombinant+fusion+proteins+using+sortase+A+and+elastin-like+polypeptides.">Three-in-one chromatography-free purification, tag removal, and site-specific modification of recombinant fusion proteins using sortase A and elastin-like polypeptides.</a> Angewandte Chemie. 52, 3703-3708 (2013).</li><li>Compton, S. J., Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+dye+response+and+interference+in+the+Bradford+protein+assay.">Mechanism of dye response and interference in the Bradford protein assay.</a> Anal Biochem. 151, 369-374 (1985).</li><li>McPherson, D. T., Xu, J., Urry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product+purification+by+reversible+phase+transition+following+Escherichia+coli+expression+of+genes+encoding+up+to+251+repeats+of+the+elastomeric+pentapeptide+GVGVP.">Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP.</a> Protein expression and purification. 7, 51-57 (1996).</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=Purification+of+recombinant+proteins+by+fusion+with+thermally-responsive+polypeptides.">Purification of recombinant proteins by fusion with thermally-responsive polypeptides.</a> Nat Biotechnol. 17, 1112-1115 (1999).</li><li>McDaniel, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+thermally+responsive+nanoparticles+of+a+genetically+encoded+peptide+polymer+by+drug+conjugation.">Self-assembly of thermally responsive nanoparticles of a genetically encoded peptide polymer by drug conjugation.</a> Angewandte Chemie. 52, 1683-1687 (2013).</li></ol>

作者没有竞争经济利益披露。

电子学习产品提供净化剥削他们的刺激 - 反应相行为的一种廉价和套色免手段。这种方法利用电子学习产品及肽或蛋白质ELP融合的LCST行为的优势在大肠杆菌的基因编码的电子学习产品的表现后，消除可溶性和不可溶性污染物大肠杆菌 。这便于纯化的，可用于生产电子学习产品用于各种应用，或可被利用为重组肽或蛋白，其中所述ELP可以作为纯化标记可与纯化后的处理来除去的纯化。 ELP净化包括预备步骤裂解大肠杆菌大肠杆菌 ，并从粗培养裂解物（ 图1），然后除去残留的可溶性和不溶性杂质的由ITC（ 图2）去除基因组和不溶性细胞碎片。由ADDI触发ELP过渡后离心热或盐tion从上清液中的可溶性污染物分离ELP，在步骤称为热自旋。 resolubilizing对ELP后，将溶液再次离心以低于T T的温度下，以除去不溶性杂质沉淀，在步骤称为冷旋。冷热交替自旋提高与每个周期的ELP溶液的纯度，以小的成本得到。纯化产量取决于ELP T T，长度，和稠合的肽或蛋白质而变化。通常情况下，该协议得到每升E的100毫克纯化ELP的大肠杆菌培养，但产率可高达500毫克/升。对ELP产品的最终纯度是通过SDS-PAGE（ 图3）确认。纯化ELP的分子量应与由ELP基因编码的理论分子量密切匹配。然而，一些电子学习产品在SDS-PAGE上迁移带的表观分子量低于其预期的分子量09 27高可达20％。电子学习产品的更精确的分析分子量可通过MALDI-TOF-MS，这也可以提供对ELP产物连同诸如高效液相色谱（HPLC）正交分析技术的纯度的附加信息来实现。 在纯化之后，在ELP T T是由程序升温比浊法测定。这种技术监视的ELP溶液的OD时的温度升高。对T t是浓度依赖性的，因此是可取的特征相关的ELP的预期应用的浓度系列。对于ELP聚物浊度轮廓呈现出对应于所述ELP过渡单个急剧增加从UNIMER到微米级聚集体（ 图4A）。对T t被定义为对应于在浊度更新的拐点的温度，精确地确定为相对于温度的OD的一阶导数的最大值。的可逆性ELP的相变是通过在OD的降低证实基线当温度降至低于T T（ 图4B）。有增加和减少温度梯度的浊度配置文件将在幅度和动力学有所不同，由于沉降ELP凝聚层和ELP再增变滞后。肽或蛋白质的ELP融合物同样表现出这种方式，其中稠合的ELP的肽或蛋白质的影响T T LCST行为。对于蛋白质的ELP融合物的过渡是可逆低于蛋白的熔融温度。而程序升温比浊法是对ELP产品，替代技术，如差示扫描量热法（DSC）的初始热特性优良的方法，也可用于测量ELP T T。 电子学习更复杂的结构表现出更复杂的热行为，也可以特征在于温度PROGRAMMED比浊法。 ELP二嵌段共聚物，例如，表现出相应的特征浊度配置文件，它们的温度触发的自组装成球形胶束在其临界胶束温度。这类ELP的二嵌段共聚物的外径通常首先增加0.1-0.5单位基线以上指示从unimers到胶束中的过渡，在此之后在OD的急剧增加（高达2.0个单位以上的基线）在较高的温度指示微米尺度的形成聚集体（ 图5A）。获得与DLS，测量的ELP组件的R H在溶液中的技术获取温度触发的自组装ELP结构的附加 ​​信息。改变R H同意与比浊法（ 图5B）测量的变化外径密切。 ELP unimers通常表现出一个R H &lt;10 nm的纳米粒子时组件表现出R H〜20-100 nm和聚集呈现出R H 17，23，29来获得。 由于ELP热性能的可调性，是由各种电子学习产品设计获得了一系列对T T S。重要的是要记住，固有的T T将影响纯化方案为每ELP，在极低或极高T T s将会需要最修改该标准协议的优化是很重要的。电子学习产品具有非常高的转变温度可能不适合净化使用这种方法。如果新颖的电子学习和肽或蛋白ELP融合物的设计可能会危及ELP的热响应，一个简单的组氨酸标签可包含用于通过固定化金属亲和层析纯化的替代。此外，对ELP序列的特性可能需要此协议的修改，如果客人残渣充电。该缓冲液pH的操作可以作为一个方法来改变ELP的总体电荷，以努力消除与污染物17的静电相互作用。此外，该协议适用于ELP融合与肽和时采取适当措施，以确保净化过程中不扰乱融合部分的活性的蛋白质的特殊情况。在整个这样的修改协议说明是为了指导电子学习产品的提纯，可能会提出这些挑战对于T T，押记，或融合的担忧。 电子学习产品由自己的LCST行为方式的净化提供了一个简单和色谱-免费的方式来净化广大的电子学习产品和肽或蛋白ELP融合在大肠杆菌中表达大肠杆菌 。这里总结了协议允许净化ØF使用的设备是最常见的生物实验室在一天之电子学习产品。电子学习产品及其融合的净化将方便，我们希望，鼓励ELP的设计在材料科学，生物技术和新医药应用的日益增长的多样性。

弹性蛋白样多肽（电子学习）的生物聚合物的重复五肽VPGXG其中X，客人残基，是任何氨基酸，除了脯氨酸组成。电子学习产品表现出较低的临界溶解温度（LCST）相变行为，使得均匀的ELP溶液将加热后分离成两相到其LCST时，它通常被称为在ELP文献1的逆转变温度（T T）。两相组成的非常稀的ELP球相和富ELP沉积物相。对ELP丰富沉积物上形成一个短的时间尺度在所述ELP链的聚集成微米尺寸的颗粒随后聚结。发生在一个范围内几摄氏度的这种行为，并且通常是可逆的，作为一种均匀的溶液，在返回到低于T 吨的温度下回收。 电子学习等，通常合成在大肠杆菌 （ 大肠杆菌 ）来回米那被连接到表达质粒的人造基因。该质粒然后转化到大肠大肠杆菌细胞系是最佳的蛋白表达。我们只使用了T7-lac启动 pET载体系统，用于在大肠杆菌中大量的各种电子学习产品中的表达大肠杆菌 ，虽然在酵母2-4，真菌5和植物6-8其它表达系统也已用于由其他研究者。许多方法存在基因重复建设ELP基因，包括递归定向连接（RDL）9，递归定向连接通过质粒重建（PRE-RDL）10，和重叠延伸滚环扩增（OERCA）11。工程师电子学习产品在基因水平的能力，能提供使用重组DNA技术与多种架构（ 例如 ，壶体，二嵌段，三嵌段等），可进一步与附加功能创建电子学习的机会肽和蛋白质。控制在基因水平也可以确保每个ELP被表达的确切长度和​​组成通过它的基因的质粒模板决定的，提供完全单分散性的生物聚合物的产品。 每个ELP的热性能取决于参数固有的生物聚合物如它的分子量（MW）和序列，以及外源性因素，包括其在溶液中的浓度和其它cosolutes，如盐的存在下进行。对ELP 12和其客体残基组成的长度为1，13，14都在ELP设计两个正交的参数，可以被用来控制对T t，其中疏水残基旅客和更长的链长度导致较低的T T s，而亲水性客人残留物和短链长导致较高的T T S。 ELP浓度成反比关系到T 吨 ，克的解决方案，其中吃ELP浓度有降低T T第12条盐的种类和浓度也影响ELP T T，其中盐的效果如下霍夫迈斯特系列15。亲液阴离子（Cl -的并在霍夫迈斯特系列更高）降低ELP T T和增加盐的浓度增强了这种效果。这些内在和外在的参数可以调整到所需要的一种ELP的具体应用目标温度范围内获得热行为。 电子学习产品的刺激-反应的行为是为应用，包括生物传感16，17，组织工程18，和药物输送19，20一个不同的范围非常有用。此外，当电子学习融合到在基因水平的肽或蛋白质，所述ELP可以作为一个简单的纯化标签，以用于重组打气的纯化提供一种廉价的分批法潮汐或蛋白质，无需层析21。修改纯化过程可确保熔合到ELP的肽或蛋白质的活性被维持。肽或蛋白质的ELP融合物可被纯化为应用，其中所述ELP标记是有用的22，23或可选地，当游离肽或蛋白质是必需的，蛋白酶识别位点可以在肽或蛋白与ELP之间被插入。除去ELP标签然后可以通过消化游离蛋白酶或蛋白酶的ELP融合，后者提供了额外的易于加工的最后一轮ELP纯化可以从目标肽或单独的ELP标记和蛋白酶ELP融合来实现蛋白质在一个单一的步骤24，25。以下协议描述了纯化过程的电子学习产品及肽或蛋白质ELP融合了其热性能的手段，并讨论了基本技术表征ELP产品的热响应。

<table border="0" cellpadding="0" cellspacing="0" width="1503">
	<tbody>
		<tr>
			<td>pET-24a(+) 
			pET-25b(+)</td>
			<td>Novagen</td>
			<td>69749-3 
			69753-3</td>
			<td>T7-lac expression vectors with resistance to kanamycin (pET-24) or ampicillin (pET-25).</td>
		</tr>
		<tr>
			<td>Ultra BL21 (DE3) competent cells</td>
			<td>Edge BioSystems</td>
			<td>45363</td>
			<td>Competent E. coli for recombinant protein expression.</td>
		</tr>
		<tr>
			<td>Terrific Broth (TB) Dry Powder Growth Media</td>
			<td>MO BIO Laboratories</td>
			<td>12105</td>
			<td>Media is reconstituted in DI H2O and autoclaved before use.</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>IPTG is reconstituted in DI H2O, sterile filtered, and added to cultures to induce enhanced expression.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) tablet</td>
			<td>Calbiochem</td>
			<td>524650</td>
			<td>These PBS tablets, when dissolved in 1 L of DI H2O, yield a 10 mM phosphate buffer with 140 mM NaCl, and 3 mM KCl with a pH of 7.4 at 25 &#176;C.</td>
		</tr>
		<tr>
			<td>Polyethyleneimine (PEI) Solution (~50% w/v)</td>
			<td>MP Biomedicals</td>
			<td>195444</td>
			<td>PEI is prepared as a 10% (w/v) solution in deionized H2O.</td>
		</tr>
		<tr>
			<td>Nalgene Oak Ridge high-speed centrifuge tubes, 50 mL</td>
			<td>Thermo Scientific</td>
			<td>3138-0050</td>
			<td>These round-bottom tubes withstand high-speed centrifugation of 30-50 ml.</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl)</td>
			<td>Thermo Scientific</td>
			<td>20491</td>
			<td>A stock solution of TCEP-HCl is prepared at 100 mM in DI H2O and adjusted to pH 7.0. For ELPs with a high cysteine content the stock solution of this reducing agent is added to the ELP pellet to reach 10 mM in H2O.</td>
		</tr>
		<tr>
			<td>Ready Gel Tris-HCL gel, 4-20% linear gradient polyacrylamide gel, 10 well, 30 &#956;l</td>
			<td>Bio-Rad Laboratories</td>
			<td>161-1105</td>
			<td>These linear gradient gels offer good visualization of ELPs with a range of MWs.</td>
		</tr>
		<tr>
			<td>Cupric chloride dihydrate (CuCl2-2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C454-500</td>
			<td>A filtered 0.5 M solution is prepared for negative staining of Tris-HCL polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>Anotop 10 syringe filter: 
			0.02 &#956;m 
			0.1 &#956;m 
			0.2 &#956;m</td>
			<td>Whatman</td>
			<td>6809-1002 
			6809-1012 
			6809-1022</td>
			<td>These 10 mm diameter syringe filters allow preparation of small volumes for DLS measurements.</td>
		</tr>
		<tr>
			<td>Millex-HV filter: 
			0.45 &#956;m</td>
			<td>EMD Millipore</td>
			<td>SLHVX13NK</td>
			<td>These 13 mm diameter syringe filters allow preparation of small volumes of solutions with large nanoparticle assemblies for DLS measurements.</td>
		</tr>
	</tbody>
</table>

non-chromatographic purification of recombinant elastin-like polypeptides and their fusions with peptides and proteins from escherichia coli

弹性蛋白样多肽，表现出较低的临界溶解温度的相变行为，现有以下特性转变温度为可溶性unimers并聚集成微米级的凝聚物高于其转变温度重复性的生物聚合物。弹性蛋白样多肽在基因水平的设计允许它们的顺序和长度，这决定了其热性能的精确控制。弹性蛋白样多肽被用在各种应用中，包括生物传感，组织工程和药物递送，其中所述ELP的转变温度和生物聚合物的体系结构可以被调整为所感兴趣的特定应用程序。此外，弹性蛋白样多肽的低临界溶解温度的相变行为允许其纯化通过其热响应，使得它们的选择性凝聚和再增允许移除可溶性和不溶性杂质的S在进行在大肠杆菌中的表达。这种方法可以用于单独使用或作为纯化的工具，肽或蛋白融合体，其中的重组多肽或蛋白的基因追加到弹性蛋白样多肽的标记可以在不色谱纯化弹性蛋白样多肽的纯化。本协议描述的弹性蛋白样多肽及其肽或融合蛋白纯化，并讨论了基本表征技术评估纯弹力蛋白样多肽产品的热性能。

在这里，我们描述了具有代表性的结果为ELP的均聚物和ELP二嵌段共聚物的纯化和鉴定。下面的表达式中的24小时的大肠杆菌大肠杆菌，细胞通过离心收集，并通过超声处理裂解。通常，在彩色微妙的变化发生在超声处理后，证实了充分的细胞裂解（ 图1A）。加入聚乙烯亚胺后，基因组组分和细胞碎片通过离心收集，分离可溶性ELP在上清液中（ 图1B）的大丸不溶性杂质。 ELP被逆转变循环（ITC），它利用了LCST行为的ELP从可溶性和不溶性杂质（ 图2）28分离进一步提纯。对ELP的相变是通过加热和/或盐的触发。离心结果形成一个小球在那个由ELP一个管的底部第二部分不溶性杂质（ 图1C）。在上清这一步，称为热自旋丢弃可溶性污染物而ELP颗粒被保留，并悬浮于冷缓冲液。该resolubilized ELP的在4℃下再离心这一步称为冷旋，丢弃不溶性杂质的颗粒，同时保留含有可溶性ELP的上清液。反复冷热交替自旋周期增加了电子学习产品的纯度，但略有减少产量，作为ELP残留在每个德祥轮离心管和移液吸头丢失的成本。 电子学习和肽或蛋白ELP融合物的成功的纯化是通过SDS-PAGE证实。在整个纯化过程中采取的小等分试样表明ELP的来自大肠杆菌的逐步纯化大肠杆菌裂解液。 ELP的过度表达在大肠杆菌粗大肠杆菌裂解物和污染物减少机智ħ连续两轮ITC（ 图3）。纯化ELP显示为接近预测的理论分子量为单一条带。 对ELP T T的特点是程序升温比浊法。一个ELP均聚物的浊度分布呈现在350纳米（约2.0个单位以上的基准为25微米的ELP浓度）单急剧增加，外径（ 图4A）。冷却浊扫描确认ELP转变的可逆性质的外径返回到基线时的温度降低低于T 吨 （ 图4B）。 ELP的嵌段共聚物表现出更复杂的浊度更新相比，均聚物电子学习。外径通常首先会增加至0.1〜0.5个单位以上的基准，在此之后，外径急剧增加至2.0个单位以上的基线（ 图5A）。 DLS测量提供了有关在R H的变化的信息相对于温度，合<html> rroborating的信息由浊度剖面（ 图5B）获得。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51583/51583fig1highres.jpg" width="600" /> 图1。初步ELP纯化后的表达，E的24小时大肠杆菌细胞通过离心收集并重新悬浮于PBS中。 甲）将细胞通过超声处理，这是伴随着在色，使得超声裂解液B）后变暗除了PEI后，冷凝的DNA污染物，溶解物是一个微妙的变化裂解离心和不溶性碎片的大的团块从可溶性ELP中分离上清液。℃）的ELP过渡诱导与另外的热量和/或盐和离心形成一个半透明的ELP粒料。ANK“&gt;请点击这里查看该图的放大版本。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51583/51583fig2highres.jpg" width="600" /> 图2。逆过渡骑自行车。ELP是由来自可溶性和不溶性杂质及其LCST行为的手段纯化。与ELP富​​含裂解物（1）所述ELP过渡诱导与另外的热量和/或盐和ELP开始通过离心（热自旋）（2）分开。对ELP粒料被保留（3a）中，同时含有水溶性污染物弃去上清液（图3b）。对ELP再悬浮于冷缓冲液（4）中，在4℃（冷旋）再次离心分离（5）。含有不溶性杂质弃去沉淀（图6a），同时保持含有可溶性ELP的上清液（图6b）。反复冷热交替自旋周期，直到所需的纯度达到，与确定SDS-PAGE电泳。 <a href="https://www.jove.com/files/ftp_upload/51583/51583fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51583/51583fig3highres.jpg" width="600" /> 的ELP净化产品图3。SDS-PAGE分析。的ELP的成功纯化通过SDS-PAGE证实。过表达ELP是显而易见的，在原油E.超声波处理后， 大肠杆菌裂解液（2）。加入的PEI和离心后的水溶性ELP的基本上保留在上清液中（3），尽管一些ELP是失去了在不溶性污染物的废弃粒料（4）。上清液以下所述第一热自旋包含显著可溶性污染物的水平，（5）。上清液以下所述第一冷旋表示ELP的不溶性杂质分离效果好（6）。热（7）和c的附加周期老自旋（8）除去残留的可溶性和不溶性杂质，分别，直到最终热（9）和冷旋（10）的上清液表现出无外来杂质带，证实了ELP产品的令人满意的纯度。这ELP均聚物，与SKGPG-（VGVPG）序列80-Y，有33.37 kDa的预期分子量。 <a href="https://www.jove.com/files/ftp_upload/51583/51583fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51583/51583fig4highres.jpg" width="600" /> 图4。ELP的均聚物通过程序升温比浊法表征。电子学习产品T T是通过监测OD在350nm处的速率1℃/ min的A）ELP均聚物表现出在一个单一的急剧增加，同时增加的温度决定外径是DEF分级表其T T在给定浓度B）所述ELP过渡的可逆性是通过监测OD为25μ摩尔溶液350nm处而降低温度，其中可逆性由OD的回归到基线确定验证。这ELP均聚物具有序列SKGPG-（VGVPG）80-Y。 <a href="https://www.jove.com/files/ftp_upload/51583/51583fig4highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51583/51583fig5highres.jpg" width="600" /> 图5。表征程序升温比浊法和动态光散射ELP的二嵌段共聚物。电子学习产品会经过纳米尺度的自组装，如ELP的二嵌段共聚物自组装成球形胶束，表现出更复杂的浊度进行访问。 <stron在OD G&gt; A）适度增加表示从unimers到胶束前，指示ELP完全聚集成微米尺寸的聚集体。 乙）DLS测量确认从一个浊度更新所推断的行为在OD快速增加的过渡，溶液在25μM的通过提供关于在R H的变化相对于温度的信息。此处所述ELP UNIMER具有R H〜8nm的和胶束具有R H〜25纳米。这ELP二嵌段共聚物具有序列GCGWPG-（VGVPG）60 - （AGVPGGGVPG）30 PGGS。 <a href="https://www.jove.com/files/ftp_upload/51583/51583fig5highres.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli at JoVE.com

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

弹性蛋白样多肽是刺激 - 反应的生物聚合物与应用，从重组蛋白纯化给药。这个协议描述了来自大肠杆菌的使用其低临界溶解温度的相变行为作为一个简单的替代色谱弹性蛋白样多肽和其肽或蛋白质融合的纯化和表征。

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

non-chromatographic-purification-recombinant-elastin-like

Research

JoVE Journal

Biology

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

21.5K 次观看.  Duke University. 弹性蛋白样多肽是刺激 - 反应的生物聚合物与应用，从重组蛋白纯化给药。这个协议描述了来自大肠杆菌的使用其低临界溶解温度的相变行为作为一个简单的替代色谱弹性蛋白样多肽和其肽或蛋白质融合的纯化和表征。 

视频: Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

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 ...

科研

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

纯化及复性的淀粉样纤维（他）<sub&gt; 6</sub&gt; -tagged重组Shadoo蛋白表达的包涵体<em&gt;大肠杆菌</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

从 大肠杆菌 和其他细菌中可扩展地分离和纯化细胞外囊泡

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...