作者感谢 m. Rubini 博士 (德国 Konstanz) 提供质粒编码 pyrrolysyl tRNA 和提供 pRSFduet pyrtRNAsynth 编码相应的氨酰 tRNA 合成酶。

1. BMP2 变型 BMP2-K3Plk 的生产
<ol><li>利用 PCR 方法对 BMP2-K3Plk 进行现场定向诱变克隆研究12<ol>
<li>用正向底漆 (5 ' GACCAGGACATATGGCTCAAGCCTAGCACAAACAGC 3 ') 和反向底漆 (5 ' CCAGGAGGATCCTTAGCGACACCCACAACCCT 3 '), 从 p25N-hmBMP2 矢量放大人类成熟 BMP2 (hmBMP2) (参见材料表), 引入琥珀色停止密码子 (标记) 在 BMP2´s 成熟部分的第一赖氨酸的位置。使用 H2O 的33.5 µL 进行 PCR 反应, 10 µL PCR 反应混合, 1.5 µL 10 µM dNTP 库存溶液, 1.5 µL 的前底漆 (10 pmol/µL), 1.5 µL 的反向底漆 (10 pmol/µL), 1 µL p25N-hmBMP2 (10 ng/µL)和1µL DNA 聚合酶 (参见材料表) 在热循环仪 (变性在95°c 为5分钟, 30 周期变性在95°c 为1分钟, 退火在60°c 为1分钟, 伸长在72°c 为1分钟和最后的引伸在72°c 为10分钟)。</li>
		<li>使用制造商推荐的商用工具包纯化 PCR 产品 (请参阅材料表)。</li>
		<li>用16µL H2O、3µL 消化缓冲液、10µL DNA (20 NdeI/µL) 和1µL 的限制酶, 以限制酶为45分钟消化 PCR 产物。热停用65°c 的酶5分钟. 用16µL h2O、3µL 消化缓冲液、10µL DNA (20 BamHI/µL) 和1µL 的限制酶, 用限制酶在37摄氏度上消化1小时的 PCR 产物。热停用酶80°c 5 分钟。</li>
		<li>使用16µL h2O、3µL 消化缓冲、10µL 的 DNA (20 ng/µL) 和1µL 的限制酶, 将 pET11a-pyrtRNA 向量与 NdeI 在37摄氏度的2小时和45分钟内消化。热停用65°c 为5分钟. 用 BamHI 37 °c 的 pET11a-pyrtRNA 向量消化 1 h, 使用16µL h2O, 3 µL 消化缓冲, 10 µL DNA (20 ng/µL), 1 µL 限制酶。热停用80摄氏度, 5 分钟。</li>
		<li>用琼脂糖凝胶电泳 (0.8% 琼脂糖, 60 分钟 100 V) 分离未消化的载体和消化残留物的消化载体。结扎在室温 (RT) BMP2-K3TAG 中消化 pET11a-pyrtRNA 骨干, 用100吴 pET11a-pyrtRNA 骨干和 34.5 ng BMP2-K3TAG 插入 (1:3 摩尔比) 进行消化后的插入。反应混合物包含 dna 骨干和插入, 2 µL 连接酶缓冲器, 1 µL 的 dna 连接酶,H2 O 到总容积20µL。</li>
		<li>在 BL21 (DE3) 细菌中进行 pET11a-pyrtRNA-BMP2-K3Plk 和 pSRF-pyrtRNAsynth 的共同转化:<ol>
<li>将每种质粒的 50 ng 添加到细菌中, 将混合物放在冰上30分钟, 热休克为42摄氏度, 五十年代, 在冰上放置5分钟, 在混合物中加入500µL 的溶源性汤 (磅) 培养基, 并在300分钟内摇动60分钟。</li>
			<li>将100µL 的细菌混合物涂在预热的卡那霉素 (50 µg/毫升)/氨苄西林 (100 µg/毫升) 板上, 并在37摄氏度一夜之间孵化。</li>
		</ol>
</li>
	</ol></li>
	<li>BMP2-K3Plk 的表达和纯化13,14<ol>
<li>在50毫升的 LB 培养基中以50µg/毫升卡那霉素和100µg/毫升氨苄西林在37摄氏度的情况下, 在一夜之间繁殖一只蜂群。</li>
		<li>稀释隔夜文化1:20 到800毫升的了不起的肉汤 (TB) 培养基补充50µg/毫升卡那霉素和100µg/毫升氨苄西林, 并增长37°c, 直到达到 OD600 0.7。添加炔丙基赖氨酸到最终浓度为10毫米。在异丙基β-D-1-thiogalactopyranoside (IPTG) 诱导前, 从培养基中收集100µL 样品。</li>
		<li>通过添加 IPTG 诱导基因表达, 最终浓度为1毫米。在 180 rpm 的轨道振动筛中, 以16小时为37摄氏度的培养。从文化中收集100µL 样本。</li>
		<li>离心机的整个文化在 9000 x g 30 分钟丢弃上清, 并并用重悬细菌颗粒在30毫升的 TBSE 缓冲 (10 毫米三, 150 毫米氯化钠, 1 毫米乙二胺乙酸 (EDTA)) 与 1:1000 (v/v) 2-巯基乙醇 (新鲜添加)。 注意事项: 在通风罩下处理2巯基乙醇。避免接触皮肤和眼睛。避免吸入蒸汽或薄雾。使用包含 2-巯基乙醇的缓冲器在通风罩下执行所有泥沙步骤。</li>
		<li>称空离心机烧杯并将悬浮颗粒转移到空烧杯中。离心机在 6360 x g 为20分钟, 丢弃上清液, 用颗粒称量烧杯。减去空烧杯的重量来计算颗粒的重量。 注意: 协议可以在这里暂停。在短期内或在-80 摄氏度的长期冷冻颗粒在-20 °c。恢复该协议后, 在 RT 中解冻颗粒。</li>
		<li>并用重悬中的颗粒 (10 毫米三 pH 8.0; 150 毫米氯化钠; 1 毫米 EDTA, 375 毫米蔗糖; 1:1000 (v/v) 2-巯基乙醇 (新鲜添加))。每10克球团使用200毫升的圣凯瑟缓冲器。</li>
		<li>油脂实验在冰上悬浮 (10 分钟以四十年代脉冲, 二十年代刹车和30% 振幅)。离心机在 6360 x g 为20分钟, 丢弃上清液并称量颗粒。重复超声波和离心步骤4次。</li>
		<li>并用重悬100毫升的 tb 缓冲器 (10 毫米三, 150 毫米氯化钠) 的颗粒。离心机在 6360 x g 为20分钟, 丢弃上清和重粒。</li>
		<li>并用重悬颗粒在核酸酶缓冲器 (100 毫米三, 1 毫米 EDTA, 3毫米氯化镁 2) 与新鲜添加 80 U/毫升核酸酶 (例如, Benzonase) 使用10毫升/克丸。在搅拌板 (30 rpm) 上在 RT 上孵化过夜。</li>
		<li>添加海卫四缓冲器 (60 毫米 EDTA, 1.5 米氯化钠, 6% (v/v) 4-(11, 33-Tetramethylbutyl) 苯基聚乙二醇) 悬浮。增加的海卫的缓冲容量对应于悬浮的0.5 容量部分。在 RT 中孵育10分钟 (不搅拌)。离心机在 6360 x g 为20分钟, 丢弃上清液并称量颗粒。</li>
		<li>并用重悬颗粒在 TE 缓冲器 (100 毫米三, 20 毫米 EDTA) 使用8毫升/克颗粒。离心机在 6360 x g 为20分钟, 丢弃上清液并称量颗粒。</li>
		<li>并用重悬颗粒加入4毫升/克25毫米 NaAc pH 5.0 和5毫升/克6米 GuCl 与1毫米 dithiothreitol (新添加)。在搅拌板 (30 rpm) 上, 在4°c 处孵化过夜。</li>
		<li>离心机在 75500 x g 20 分钟。上清液现在包含展开的单体 BMP2 蛋白。收集上清, 并把这个提取物浓缩到 20 OD/毫升使用 3 kDa 分子量切断 (截留分子量) 膜在浓缩细胞 (见材料表)。</li>
		<li>将浓缩单体加入复缓冲液 (2 米 LiCl, 50 毫米三, 25 毫米, 5 毫米 EDTA, 1 毫米谷胱甘肽 (GSSG), 2 毫米谷胱甘肽 (GSH)) 搅拌 (30 rpm)。在黑暗中在 RT 中孵化120小时。 注意: 在此潜伏期内, 复 occours。从这个步骤开始的解决方案包含折叠二聚体 BMP2-K3Plk。</li>
		<li>使用浓缩的 hcl 将溶液的 pH 值调整为 3.0. 透析解决方案对1毫米 hcl. 用 10 kDa 分子量切断 (截留分子量) 膜在浓缩细胞中浓缩透析溶液。</li>
		<li>通过添加缓冲 A (20 毫米 NaAc, 30% 异丙醇) 平衡解决方案。添加与解决方案的30% 卷对应的缓冲区容量。离心溶液在 4700 x g 15 分钟。</li>
		<li>平衡在快速蛋白质液相色谱 (FPLC) 系统上进行离子交换的柱状体15与150毫升缓冲器 a. 将蛋白质溶液加载到平衡柱上。使用100毫升的缓冲器 b (20 毫米 NaAc, 30% 异丙醇, 2 米氯化钠) 的线性梯度洗脱分数 b. 收集2毫升的分数。 注: 根据总柱容积将蛋白质按入列前浓缩。最后的蛋白质体积应 < 总柱容积的5%。</li>
		<li>通过 sds 聚丙烯酰胺凝胶 (12%) 电泳 (sds 页) 16和考马斯亮蓝染色17 (参见材料表) 分析了每一个分数的20µL。 注: 考马斯亮蓝色染色 SDS 页凝胶显示含有脂肪酸 (26 kDa) 和含有单体的分数 (13 kDa) 的分数。</li>
		<li>池中含有二聚体的分数。透析含馏分的二聚体池, 隔夜在4°c 对1毫米 HCl (5 公升容量)。使用 10 kDa 浓缩离心过滤装置集中最终产品 (请参阅材料表)。用 sds 聚丙烯酰胺 (12%) 凝胶电泳 (sds 页) 和考马斯蓝染色法对最终产物进行分析。 注: 考马斯亮蓝染色的 SDS 页凝胶上的带子应该只显示一个波段在 26 kDa。这是最后的 BMP2-K3Plk 产品。</li>
	</ol></li>
</ol>2. 优化铜 (I)-催化炔烃-叠氮化物 Cycloaddition (CuAAC) 条件
<ol><li>抗坏血酸钠 (NaAsc) 和铜 (II) 硫酸盐 (丘索4 )对野生型 BMP2 (BMP2-WT)的影响<ol>
<li>孵化20µM BMP2-WT 与 NaAsc 的不同比率丘索4。在50µL 卷中使用 0.5 mm NaAsc 和 0.5 mm 丘索4 (起始浓度)。继续以下 NaAsc 到丘索4: (1:1), (1.7: 1), (10:1), (20: 1), 保持丘索4浓度常数在0.5 毫米和调整 NaAsc 的浓度相应。在 RT 上一夜之间在旋转搅拌机 (20 rpm) 上执行 H2O 的反应。</li>
		<li>通过添加含有 5% 2-巯基乙醇和孵育样品的载入缓冲剂, 在95摄氏度进行6分钟, 制备减少的样品. 用 sds 聚丙烯酰胺凝胶 (12%) 电泳 (sds 页) 和考马斯蓝染色法分析减少和不减少的样品。Coomasie 染色的 SDS 页凝胶显示带在 13 kDa (减少条件) 到 26 kDa 和更高的分子量 (不减少的条件) 的范围。</li>
	</ol>
</li>
	<li>
三 (3-hydroxypropyltriazolylmethyl) 胺 (THPTA) 对 CuAAC 反应的影响
	<ol>
<li>孵化20µM BMP2-K3Plk 与 THPTA 的不同比率丘索4。使用5毫米 NaAsc 和200µM 3-叠氮基 7-hydroxycoumarin (保持 NaAsc 和 3-叠氮基-7-hydroxycoumarin 常数)。使用50µM THPTA 和0.5 毫米的丘索4作为起始浓度。使用 THPTA 与丘索的不同比率4: (7:1);(10:1);(12:1);(15:1);(20:1). 在 H2O (50 µL 总容积) 中对旋转混合器 (20 rpm) 上的 RT 执行 24 h 的反应。</li>
		<li>在联轴器上, 3-叠氮基-7-hydroxycoumarin 成为荧光染料。在减少和不减少的条件下准备样品, 并通过 SDS 页面进行分析。用特定波长为 3-叠氮基-7-hydroxycoumarin (λ abs = 404 毫微米;λ em = 477 nm) 在激发通道下可视化凝胶。</li>
	</ol>
</li>
</ol>3. BMP2-K3Plk 对叠氮化物功能性琼脂糖珠的共价耦合技术
<ol><li>孵育20µM 的 BMP2-K3Plk 或 BMP2-WT (用作负控制) 与20µL 的叠氮化物活化琼脂糖珠在总容积500µL 的反应缓冲 (0.1 米 HEPES pH 7.0, 3.9 米尿素, 50 µM 丘索4, 250 µM THPTA, 5 毫米抗坏血酸钠) 为 2 h 在 RT 在 rotating 搅拌机 (20 rpm)。通过添加5毫米 EDTA (最终浓度) 停止反应。</li>
	<li>在旋转搅拌机上孵育15分钟 (20 rpm)。离心机样品在 2万 x g 为1分钟在 RT. 收集上清液。</li>
	<li>用三倍于哈佛商学院500缓冲器 (50 毫米 HEPES, 500 毫米 NaCl), 三倍, 1000 µL 4 米氯化镁2和两次与1000µL 磷酸盐缓冲盐水 (PBS) 清洗含有珠子的球团, µL 1000。在每一个洗涤步骤, 离心机在 2万 x g 1 分钟的 RT 和收集上清液。在1000µL 的 PBS 中存储耦合珠, 4 摄氏度。 注意: 清除上清液时请勿扰乱珠粒。</li>
</ol>4. 使用德州红标记的 BMP 受体 I A 胞外区 ( BMPR欧共体) 验证固定化 BMP2-K3Plk 的存在和生物活性
<ol><li>孵化50µL 的 BMP2-K3Plk 功能性珠子与1µM 德州红标记 BMPR IAEC与150µL 的哈佛商学院500缓冲器 (50 毫米 HEPES, 500 毫米 NaCl) 在 RT 为 1 h 在旋转搅拌机 (20 rpm)。</li>
	<li>将珠子离心 2万 x 克, 在 RT 上1分钟. 丢弃上清。用1000µL 的哈佛商学院500缓冲区来清洗珠子。重复洗涤步骤额外的3次。每洗一步后, 2万 x 克离心机在 RT 1 分钟。</li>
	<li>并用重悬在500µL 的 PBS 和吸管50µL 在玻璃封面幻灯片的珠子。将显微镜过滤器设置在561或 594 nm 之间。检测德州 Red–BMPREC-BMP2-K3Plk 功能化珠子使用荧光显微镜和拍照。</li>
</ol>5. 测定碱性磷酸酶 (磷酸) 表达式, 以证明在偶联反应前后产生的 BMP2-K3Plk 的体外生物活性。
<ol><li>
碱性磷酸酶 (ALP) 测定法
	<ol>
<li>培养 promyoblastic C2C12 细胞 (ATCC CRL-172) 在 Dulbecco 的改良鹰的培养基 (DMEM) 与10% 胎小牛血清 (FCS), 100 U/毫升青霉素 G 和100µg/毫升链霉素在37°c 在湿润气氛 5% CO2。</li>
		<li>种子 C2C12 细胞在密度为 3 x 104细胞/以及到96井微板块。让细胞整夜附着。在 100 CO100 37 h 的湿润气氛中, 去除培养基和孵化 C2C12 细胞, 在100µL/井 DMEM (2% FCS, 5% U/毫升青霉素 G 和2µg/毫升链霉素) 存在 0.5-200 nM BMP2-WT 或 BMP2-K3Plk 在72摄氏度。</li>
		<li>用100µL 的 PBS 去除培养基和洗涤单元。溶解细胞在 RT 1 小时与100µL/井溶解缓冲 (1% NP-40, 0.1 米甘氨酸, pH 9.6, 1 毫米氯化镁2, 1 毫米 ZnCl2) 在震动板 (220 rpm)。</li>
		<li>添加100µL/井的磷酸酶缓冲 (0.1 米甘氨酸, pH 9.6, 1 毫米氯化镁2, 1 毫米 ZnCl2) 与1毫克/毫升 p 硝基苯磷酸 (新鲜添加) 到细胞裂解。</li>
		<li>测量吸收 405 nm 在 multiplate 读取器添加的碱性磷酸酶缓冲, 并每5分钟, 直到颜色的发展完成。使用逻辑模型生成剂量响应曲线和 EC50 值, y = a2 + (12)/(1 + (x/x0)p)。</li>
	</ol>
</li>
	<li>
碱性磷酸酶 (磷酸) 染色
	<ol>
<li>培养 promyoblastic C2C12 细胞 (ATCC CRL-172) 在 Dulbecco 的改良鹰培养基 (DMEM) 与10% 胎小牛血清 (FCS), 100 U/毫升青霉素 G 和100µg/毫升链霉素在37°c 的湿润气氛在 5% CO2。</li>
		<li>种子 C2C12 细胞在密度为 3 x 104细胞/以及在96井微板块。让细胞整夜附着。移除介质并添加20µL/井 BMP2-K3Plk 耦合珠。BMP2-K3Plk 耦合珠是在1000年µL 的 PBS 悬浮。</li>
		<li>在 DMEM 中制备了0.4% 低熔点琼脂糖溶液。熔化在微波 (600 W 为3分钟) 并且让它凉快在水浴在37°c 直到使用。</li>
		<li>在每个井中添加20µL 0.4% 低熔点琼脂糖 (覆盖珠和细胞)。离心机在 2000 x g 5 分钟在20摄氏度。添加80µL DMEM (2% FCS, 100 U/毫升青霉素 G 和100µg/毫升链霉素) 和孵化在37摄氏度的湿润气氛在 5% CO2 72 h。</li>
		<li>去除培养基, 小心不要分离凝固的0.4% 琼脂糖。添加100µL/井1步 NBT/BCIP (硝基蓝四氮唑氯化物, 5-溴-4-氯-3 '-indolyphosphate 对甲苯胺盐) 基质溶液。</li>
		<li>在紫色染色后立即使用光镜分析碱性磷酸酶染色。使用明亮的现场显微镜和拍照。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Giannoudis, P. V., Dinopoulos, H., Tsiridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+substitutes:+An+update.">Bone substitutes: An update.</a> Injury. 36, S20-S27 (2005).</li><li>Oryan, A., Alidadi, S., Moshiri, A., Bigham-Sadegh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+proteins:+A+powerful+osteoinductive+compound+with+non-negligible+side+effects+and+limitations.">Bone morphogenetic proteins: A powerful osteoinductive compound with non-negligible side effects and limitations.</a> Biofactors. 40 (5), 459-481 (2014).</li><li>Luginbuehl, V., Meinel, L., Merkle, H. P., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+delivery+of+growth+factors+for+bone+repair.">Localized delivery of growth factors for bone repair.</a> Eur J Pharm Biopharm. 58 (2), 197-208 (2004).</li><li>Haidar, Z. S., Hamdy, R. C., Tabrizian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+recombinant+bone+morphogenetic+proteins+for+bone+regeneration+and+repair.+Part+A:+Current+challenges+in+BMP+delivery.">Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part A: Current challenges in BMP delivery.</a> Biotechnol Lett. 31 (12), 1817-1824 (2009).</li><li>Hollinger, J. O., Uludag, H., Winn, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+release+emphasizing+recombinant+human+bone+morphogenetic+protein-2.">Sustained release emphasizing recombinant human bone morphogenetic protein-2.</a> Adv Drug Deliv Rev. 31 (3), 303-318 (1998).</li><li>Uludag, H., 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=Implantation+of+recombinant+human+bone+morphogenetic+proteins+with+biomaterial+carriers:+A+correlation+between+protein+pharmacokinetics+and+osteoinduction+in+the+rat+ectopic+model.">Implantation of recombinant human bone morphogenetic proteins with biomaterial carriers: A correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model.</a> J Biomed Mater Res. 50 (2), 227-238 (2000).</li><li>Uludag, H., Golden, J., Palmer, R., Wozney, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinated+bone+morphogenetic+protein-2:+In+vivo+and+in+vitro+activity.">Biotinated bone morphogenetic protein-2: In vivo and in vitro activity.</a> Biotechnol Bioeng. 65 (6), 668-672 (1999).</li><li>Aono, 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=Potent+ectopic+bone-inducing+activity+of+bone+morphogenetic+protein-4/7+heterodimer.">Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer.</a> Biochem Biophys Res Commun. 210 (3), 670-677 (1995).</li><li>Suzuki, 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=Alginate+hydrogel+linked+with+synthetic+oligopeptide+derived+from+BMP-2+allows+ectopic+osteoinduction+in+vivo.">Alginate hydrogel linked with synthetic oligopeptide derived from BMP-2 allows ectopic osteoinduction in vivo.</a> J Biomed Mater Res. 50 (3), 405-409 (2000).</li><li>Knaus, P., Sebald, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperativity+of+binding+epitopes+and+receptor+chains+in+the+BMP/TGFbeta+superfamily.">Cooperativity of binding epitopes and receptor chains in the BMP/TGFbeta superfamily.</a> Biol Chem. 382 (8), 1189-1195 (2001).</li><li>Wang, L., Xie, J., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+genetic+code.">Expanding the genetic code.</a> Annu Rev Biophys Biomol Struct. 35, 225-249 (2006).</li><li>Costa, G. L., Weiner, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+PCR+site-directed+mutagenesis.">Rapid PCR site-directed mutagenesis.</a> CSH Protoc. 2006 (1), (2006).</li><li>Kirsch, T., Nickel, J., Sebald, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+recombinant+BMP+receptor+IA+ectodomain+and+its+2:1+complex+with+BMP-2.">Isolation of recombinant BMP receptor IA ectodomain and its 2:1 complex with BMP-2.</a> FEBS Lett. 468 (2-3), 215-219 (2000).</li><li>Tabisz, B., 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=Site-directed+immobilization+of+BMP-2:+Two+approaches+for+the+production+of+innovative+osteoinductive+scaffolds.">Site-directed immobilization of BMP-2: Two approaches for the production of innovative osteoinductive scaffolds.</a> Biomacromolecules. 18 (3), 695-708 (2017).</li><li>Duong-Ly, K. C., Gabelli, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+ion+exchange+chromatography+to+purify+a+recombinantly+expressed+protein.">Using ion exchange chromatography to purify a recombinantly expressed protein.</a> Methods Enzymol. 541, 95-103 (2014).</li><li>Brunelle, J. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-dimensional+SDS-polyacrylamide+gel+electrophoresis+(1D+SDS-PAGE).">One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).</a> Methods Enzymol. 541, 151-159 (2014).</li><li>Brunelle, J. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coomassie+blue+staining.">Coomassie blue staining.</a> Methods Enzymol. 541, 161-167 (2014).</li><li>Kirsch, T., Nickel, J., Sebald, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP-2+antagonists+emerge+from+alterations+in+the+low-affinity+binding+epitope+for+receptor+BMPR-II.">BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II.</a> EMBO J. 19 (13), 3314-3324 (2000).</li><li>Hein, J. E., Fokin, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-catalyzed+azide-alkyne+cycloaddition+(CuAAC)+and+beyond:+new+reactivity+of+copper(I)+acetylides.">Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and beyond: new reactivity of copper(I) acetylides.</a> Chem Soc Rev. 39 (4), 1302-1315 (2010).</li><li>Alborzinia, H., 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=Quantitative+kinetics+analysis+of+BMP2+uptake+into+cells+and+its+modulation+by+BMP+antagonists.">Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists.</a> J Cell Sci. 126 (Pt 1), 117-127 (2013).</li><li>Paarmann, P., 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=Dynamin-dependent+endocytosis+of+Bone+Morphogenetic+Protein2+(BMP2)+and+its+receptors+is+dispensable+for+the+initiation+of+Smad+signaling.">Dynamin-dependent endocytosis of Bone Morphogenetic Protein2 (BMP2) and its receptors is dispensable for the initiation of Smad signaling.</a> Int J Biochem Cell Biol. 76, 51-63 (2016).</li><li>Pohl, T. L., Boergermann, J. H., Schwaerzer, G. K., Knaus, P., Cavalcanti-Adam, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+immobilization+of+bone+morphogenetic+protein+2+via+a+self-assembled+monolayer+formation+induces+cell+differentiation.">Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation.</a> Acta Biomater. 8 (2), 772-780 (2012).</li></ol>

作者声明没有竞争的财政利益。

通过遗传密码子扩展生成标记的蛋白质变体允许在主要蛋白质序列的任何位置引入各种非天然氨基酸类似物。如果像 BMP2 这样的 BMPs, 常见的标签, 如 6-组氨酸 (他) 的标签只能被引入 n-末期, 因为 protein´s 的 C 末端是埋在三级蛋白质结构, 因此不能从外部进入。在其他位置, 引入标签的大小很可能会导致结构的改变, 从而抹杀 BMP´s 生物活性。此外, 引入突变的 BMP2 序列可以影响复性的功效, 也可以改变其他蛋白质参数, 如等电点的修饰蛋白。因此, 建立的蛋白质生产协议中的每一步都可能需要根据改变的蛋白质 variant´s 特性进行调整。BMP2-K3Plk 的生产确实需要修改已建立的方法来表达野生型 BMP2。对不同的培养时间或炔丙基赖氨酸 (保良局朱正贤) 浓度进行了测试 (未显示数据), 以达到最高的表达率。复用, 分离和纯化步骤幸运地不需要进一步 adaptions。我们已经报告, 如果在我们的实验室生产的其他 BMP2 变种, 一些蛋白质特性发生了显著变化, 这需要修改 BMP 生产方法的几个步骤18。所生产的 BMP2-K3Plk 显示生物活性与野生型蛋白相当。差异发生在高 BMP2 浓度, 可能是由于较低的溶解度的 BMP2-K3Plk 变异在培养基相比, 野生型 BMP2。
尽管铜催化叠氮化物-炔烃 cycloaddition (CuAAC)19的已知优势, CuAAC 反应应小心地适应特定的应用。我们展示了 BMP2 是如何被分散或产生聚集或多聚体由于强烈的还原反应条件。因此, 所有反应参数必须详细分析, 以找出条件, 使蛋白质不受影响。
我们的方法, 以共价键夫妇的 BMP2 变异叠氮化物功能性琼脂糖珠通过点击化学可以实现高效, 使功能性珠触发成骨分化在体外。将野生型 BMP2 蛋白用于与珠子的反应时, 只能观察到碱性磷酸酶表达的弱染色, 表明偶联在 BMP2-K3Plk 的炔丙基噬菌体残留物中确实发生了高度的特异性。
这种位置耦合特异性反映了一个伟大的改善与固定程序涉及经典的 NHS/化学。在这种情况下, 发生随机耦合, 主要涉及赖氨酸残留物中存在的主要胺类。由于可能的连接的多样性与支架, 导致蛋白质的不同的取向对细胞感受器官, 被结合的蛋白质有一个可变的成骨活动。
使用现场直接固定化 BMP2 可能克服与高剂量可溶性 BMP2 有关的缺点, 这是需要诱导骨形成。此外, 结果清楚地表明, 某些细胞反应, 如 C2C12 细胞的成骨分化, 是完全由固定化的 BMP2 启动, 尽管最近的研究声称, 信号转导需要吞的配体/配体-受体复合体20,21。我们的研究结果与其他研究相一致, 证明 BMP2 (但不是以站点为导向) 到非 endocytosable 表面, 仍然能够诱导成骨细胞分化为22。
考虑到在偶联反应中使用了 BMP2 的超生理浓度, 可进一步降低固定化 BMP2 的用量, 同时仍能保存珠的成骨性质。然而, 在这种优化步骤之前, BMP2-K3Plk-functionalized 脚手架需要在动物实验中进行测试, 以证明其成骨潜能在体内。

骨组织工程和骨再生的最终目的是克服常规治疗非愈合骨折的缺点和局限性。自动或异体移植主要用作当前的治疗策略, 尽管它们都有几个缺点。理想的植骨应由诱导成骨和 osteoconduction 诱导成骨, 导致移植 osteointegration 骨。目前, 只有自动移植被认为是 "金标准", 因为它提供了理想的骨移植的所有特点。不幸的是, 它还提出了一些重要的负面方面, 例如长时间的手术时间和第二个创伤部位, 通常需要更多的并发症 (例如, 慢性疼痛, 血肿形成, 感染, 美容缺陷,等等)。同种异体移植, 另一方面具有所有一般方面的次优特征1。在过去几年中, 替代植骨技术得到了改善, 目的是生产骨诱导、osteoconductive、生物相容性和 bioresorbable 的支架。由于许多生物材料不显示所有这些成骨特征, 不同的生长因子, 主要 BMP2 和 BMP7, 已被纳入, 以改善其成骨潜能的特殊支架2。
作为一个基本的标准, 这种生长因子传递系统应提供一个控制剂量释放一段时间, 以促进必要的事件, 如细胞的招募和依恋, 细胞肉芽和血管生成。然而, BMPs 和其他成骨生长因子通常是固定的非共价键的3。诱捕和吸附技术需要使用超生理量的蛋白质, 由于最初的爆裂释放, 导致严重的缺点在体内,通常影响周围组织通过诱导骨过度增长,骨溶解, 肿胀和炎症4。因此, 可通过共价固定化方法, 在交付部位保留较长时间的生长因子。经过化学修饰的 BMP2 (琥珀酰化5、乙酰6 或生物素化 7)、工程化 heterodimers 8 或BMP2派生寡肽9已设计并用于克服与吸收.然而, 这些构造的生物活性是不可预知的, 因为这种安排有可能抑制固定配体对细胞受体的束缚。如前所示, 所有四受体链参与形成激活配体-受体配合物与固定化 BMP2 互动, 以充分激活所有下游信号瀑布10。
为了克服由于固定化因子的生物活性、稳定性和生物利用度等因素而产生的不均匀产品结果的问题, 我们设计了一种能以站点为导向的共价键结合支架的 BMP 变体。这个变种称为 BMP2-K3Plk, 包括由遗传密码子扩展11引入的人工氨基酸。该变种已成功地链接到脚手架使用共价键耦合战略, 同时保持其生物活性。

<table border="0" cellpadding="0" cellspacing="0" style="width:1137px;" width="1138">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Material</td>
			<td style="width:203px;"></td>
			<td style="width:136px;"></td>
			<td style="width:403px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Step NBT/BCIP</td>
			<td style="width:203px;">Thermo Fisher</td>
			<td style="width:136px;">34042</td>
			<td style="width:403px;">Add solution to cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Azido-7-hydroxycoumarin</td>
			<td style="width:203px;">BaseClick</td>
			<td style="width:136px;">BCFA-047-1</td>
			<td style="width:403px;">Chemical used for click reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Agarose low melting point</td>
			<td style="width:203px;">Biozym</td>
			<td style="width:136px;">840101</td>
			<td>Agarose for ALP assay&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Azide agarose beads</td>
			<td style="width:203px;">Jena Bioscience</td>
			<td style="width:136px;">CLK-1038-2</td>
			<td style="width:403px;">Beads used for reaction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI (Fast Digest enzyme)</td>
			<td>Thermo Fisher Scientific</td>
			<td>FD0054</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:396px;">BMP receptor IA (BMPR-IAEC)</td>
			<td>--</td>
			<td>--</td>
			<td>Produced in our lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Coomassie Brilliant Blue G-250 Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:136px;">20279</td>
			<td>Chemical used for Coomassie Brilliant blue staining of SDS PAGE</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:396px;">Copper (II) sulfate anhydrous (CuSO4)</td>
			<td style="width:203px;">Alfa Aesar</td>
			<td style="width:136px;">A13986</td>
			<td style="width:403px;">Chemical used for click reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">DNA Polymerase and reaction buffer&#160;</td>
			<td>Kapabiosystems</td>
			<td>KK2102</td>
			<td>KAPA HiFi PCR Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) GlutaMAX</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:136px;">61965-026</td>
			<td style="width:403px;">Cell culture media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:203px;">Sigma Aldrich GmbH</td>
			<td style="width:136px;">E5134-1kg</td>
			<td style="width:403px;">Chemical used to stop click reaction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Isopropyl &#223;-D-1-thiogalactopyranoside (IPTG)</td>
			<td style="width:203px;">Carl Roth GmbH</td>
			<td style="width:136px;">2316.5</td>
			<td>Bacteria induction (1mM final concentration)&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NdeI (Fast Digest enzyme)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER0581</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">NHS-activated Texas Red</td>
			<td style="width:203px;">Life technologies</td>
			<td style="width:136px;">T6134</td>
			<td style="width:403px;">Coupled to receptor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">P- Nitrophenyl Phosphate</td>
			<td style="width:203px;">Sigma Aldrich GmbH</td>
			<td style="width:136px;">N4645-1G</td>
			<td style="width:403px;">Alkaline Phosphatase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">p25N-hmBMP2&#160;</td>
			<td>--</td>
			<td>--</td>
			<td>Plasmid kindly provided from Walter Sebald to J. Nickel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET11a-pyrtRNA</td>
			<td>--</td>
			<td>--</td>
			<td>Provided by the Chair for Pharmaceutics and Biopharmacy, University Wuerzburg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">propargyl-L-lysine (Plk)</td>
			<td>--</td>
			<td>--</td>
			<td>Provided by the Chair for Pharmaceutics and Biopharmacy, University Wuerzburg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pSRFduet-pyrtRNAsynth</td>
			<td>--</td>
			<td>--</td>
			<td>Provided by the Chair for Pharmaceutics and Biopharmacy, University Wuerzburg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Qiagen Gel Extraction Kit</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:136px;">28704</td>
			<td>Gel Purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Qiagen PCR purification Kit</td>
			<td style="width:203px;">Qiagen</td>
			<td>28104</td>
			<td style="width:403px;">PCR Purification&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Sodium L-ascorbate</td>
			<td style="width:203px;">Sigma Aldrich GmbH</td>
			<td style="width:136px;">A7631-100G</td>
			<td style="width:403px;">Chemical used for click reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">T4 DNA Ligase</td>
			<td style="width:203px;">ThermoScientific</td>
			<td style="width:136px;">EL0011</td>
			<td>Ligation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">tris(3-hydroxypropyltriazolylmethyl)amine (THPTA)</td>
			<td style="width:203px;">BaseClick</td>
			<td style="width:136px;">BCMI-006-100</td>
			<td style="width:403px;">Chemical used for click reaction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol</td>
			<td style="width:203px;">Sigma Aldrich GmbH</td>
			<td>X100-1L</td>
			<td style="width:403px;">Triton X 100&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Name</td>
			<td style="width:203px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Equipment</td>
			<td style="width:203px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Amicon concentrating cell 400 ml&#160;</td>
			<td style="width:203px;">Merck KGaA</td>
			<td style="width:136px;">UFSC40001</td>
			<td style="width:403px;">Concentrating unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15 Centrifugal Filter Units</td>
			<td style="width:203px;">Merck KGaA</td>
			<td style="width:136px;">UFC901024</td>
			<td style="width:403px;">Concentrating centrifugal unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#196;KTA avant FPLC</td>
			<td>&#196;KTA</td>
			<td>--</td>
			<td>FPLC machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Avanti J-26XP</td>
			<td style="width:203px;">Beckman Coulter&#160;</td>
			<td style="width:136px;">393124</td>
			<td>Centrifuge for bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Bacterial Shaking Incubator</td>
			<td style="width:203px;">Infors HT</td>
			<td style="width:136px;"></td>
			<td>Shaking incubator for bacterial culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">FluorChem Q system</td>
			<td style="width:203px;">proteinsimple</td>
			<td style="width:136px;">--</td>
			<td>Imaging and analysis system for SDS-PAGE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Fluorescent miscroscope</td>
			<td style="width:203px;">Keyence</td>
			<td style="width:136px;">BZ-9000 (BIOREVO)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fractogel&#174; EMD SO3- (M)</td>
			<td style="width:203px;">Merck KGaA</td>
			<td style="width:136px;">116882</td>
			<td style="width:403px;">Ion Exchange Chromatography column material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Greiner CELLSTAR&#174;&#160;96 well plates</td>
			<td style="width:203px;">Sigma</td>
			<td>M5811-40EA</td>
			<td>96 well plates for cell culture (ALP Assay)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Heraeus Multifuge X1R</td>
			<td style="width:203px;">ThermoScientific</td>
			<td style="width:136px;">--</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">M-20 Microplate Swinging Bucket Rotor</td>
			<td>ThermoScientific</td>
			<td style="width:136px;">75003624</td>
			<td>Rotor for Microcentrifuge for plate during ALP staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Microcentrifuge - 5417R</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:136px;">--</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OriginPro 9.1 G&#160;</td>
			<td style="width:203px;">OriginLab</td>
			<td style="width:136px;">--</td>
			<td>software for stastic analysis of ALP assay data</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polysine Slides</td>
			<td>ThermoScientific</td>
			<td>10143265</td>
			<td>microscope slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Rotor JA-10</td>
			<td style="width:203px;">Beckman Coulter&#160;</td>
			<td style="width:136px;">--</td>
			<td>rotor for Avanti J-26XP centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Rotor JLA 8.1</td>
			<td style="width:203px;">Beckman Coulter&#160;</td>
			<td style="width:136px;">--</td>
			<td>rotor for Avanti J-26XP centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Rotor JA 25.50</td>
			<td style="width:203px;">Beckman Coulter&#160;</td>
			<td style="width:136px;">--</td>
			<td>rotor for Avanti J-26XP centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tecan infinite M200 multiplate reader</td>
			<td style="width:203px;">Tecan Deutschland GmbH</td>
			<td style="width:136px;">--</td>
			<td>Multiplate reader for ALP assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Thermocycler - Labcycler Gradient</td>
			<td style="width:203px;">SensoQuest GmbH</td>
			<td style="width:136px;">--</td>
			<td>PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">TxRed - microscope filter</td>
			<td style="width:203px;">Keyence</td>
			<td style="width:136px;"></td>
			<td>Filter for fluorescent microscope&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Ultrafiltration regenerated cellulose discs 3 kDa</td>
			<td style="width:203px;">Merck KGaA</td>
			<td style="width:136px;">PLBC04310</td>
			<td style="width:403px;">used with amicon concentrating cell 400ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Ultrafiltration regenerated cellulose discs 10 kDa</td>
			<td style="width:203px;">Merck KGaA</td>
			<td style="width:136px;">PLGC04310</td>
			<td style="width:403px;">used with amicon concentrating cell 400ml</td>
		</tr>
	</tbody>
</table>

site-directed immobilization of bone morphogenetic protein 2 to solid surfaces by click chemistry

深入探讨了不同治疗方法对骨缺损不愈合的处理策略。目前使用的治疗存在一些限制, 导致使用生物材料结合成骨生长因子, 如骨形态发生蛋白 (BMPs)。通常使用的吸收或封装方法需要超生理量的 BMP2, 通常导致一种所谓的初始爆裂释放效应, 引发了一些严重的不良副作用。解决这些问题的可能策略是将蛋白质与支架进行共价键。此外, 耦合应以特定于站点的方式进行, 以保证产品的可重现性。因此, 我们创造了一个 BMP2 变种, 其中一个人工氨基酸 (炔丙基赖氨酸) 引入到成熟的部分 BMP2 蛋白由密码子使用扩展 (BMP2-K3Plk)。BMP2-K3Plk 通过铜催化叠氮化物-炔烃 cycloaddition (CuAAC) 耦合到功能化珠。结合 BMP2-K3Plk 的生物活性被证实为体外, BMP2-K3Plk-functionalized 珠的成骨活性在细胞基检测中得到证实。与 C2C12 细胞接触的功能性珠子能够在局部受限的珠子附近诱导碱性磷酸酶 (ALP) 的表达。因此, 通过这种技术, 可以产生功能化的支架, 可以触发细胞分化为成骨的血统。另外, 由于现场定向耦合 BMP2 的控制方向, 降低 BMP2 剂量是足够的。用这种方法, BMPs 总是暴露在细胞表面的适当方向的受体, 这不是情况下, 如果通过非现场定向耦合技术耦合因素。产品的结果是高度可控的, 从而导致材料的均匀性, 提高其适用性, 修复严重大小骨缺损。

在本文中, 我们描述了一种方法, 以共价键夫妇一个新的 BMP2 变体, BMP2-K3Plk, 商业上可利用的叠氮化物功能琼脂糖珠 (图 1)。通过诱导 C2C12 细胞碱性磷酸酶 (ALP) 基因表达, 验证了 BMP2-K3Plk 变异的生物活性。体外测试显示了由野生类型 BMP2 (BMP2-WT) 和 BMP2-K3Plk (图 2) 引起的类似的 ALP 表达式级别。
铜 (II.) 硫酸盐 (丘索4) 和抗坏血酸钠 (NaAsc) 之间的氧化还原反应产生的活性氧种类可能影响结构完整性, 从而影响 BMP2-K3Plk 的生物活性。为了验证蛋白质的结构完整性, 我们进行了一夜孵化与丘索4和 NaAsc, 显示 BMP2-WT 退化以浓度依赖性方式 (由 SDS 页在减少的情况下形象化 (图 3A1)) 和在大约 40 kDa (在不减少的条件下) 可见的多聚体或聚合体的形成(图 3A 2).为避免蛋白质降解, 三 (3-hydroxypropyltriazolyl 甲基) 胺 (THPTA) 被用作保护剂 (图 3B)。使用 THPTA 防止 BMP2 破碎, 而更高分子量结构的形成不能通过添加 THPTA 来防止。反应的进一步改善处理了反应缓冲物的构成, 反应温度和反应时间 (没有显示的数据)。此处描述的协议详细介绍了反应缓冲成分、温度和反应时间等方面的最终成果。
为了证实 BMP2-K3Plk 在偶联后保留生物活性, 我们使用了 I 型受体的荧光标记受体胞外区蛋白 (BMPRIAEC) 来证明固定化蛋白仍能与此受体结合。用染料标记的 BMPREC (图 4) 孵化时, 通过 CuAAC 化学 BMP2-K3Plk 的珠子产生了荧光。与此相反, 与 BMP2-WT 一起孵化的未涂覆的珠子或珠子没有显示出背景水平以上的荧光信号 (未显示数据)14。
在确认固定配体的受体结合能力在体外, 我们还测试了生物反应是否可以触发的功能性珠在细胞的检测。碱性磷酸酶介导的染色仅发生在与 BMP2-K3Plk-functionalized 珠直接接触的细胞中 (图 5)。这证实了蛋白质确实是共价键链接到珠, 而不是仅仅吸收, 因为更广泛的染色, 在更大的距离, 珠子会被观察到。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56616/56616fig1.jpg"> 图 1: BMP2-K3Plk 的描述 (A)BMP2-K3Plk 变异的描述与介绍的氨基酸替换的本地化。(B)耦合方案: BMP2-K3Plk CuAAC 与叠氮化物功能性珠子的反应。转载 (改编), 并允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare.jove.com/files/ftp_upload/56616/56616fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56616/56616fig2.jpg"> 图 2: BMP2-variants 的生物活性.BMP2-K3Plk 与 wildtype BMP2 (BMP2-WT) 生物活性 (磷酸酶测定) 的比较。x 轴表示测定中使用的 BMP2-WT 或 BMP2-K3Plk 的浓度。EC50 数据代表4个单独实验 (N=4) 的平均值和标准偏差。转载 (改编), 并允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare.jove.com/files/ftp_upload/56616/56616fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56616/56616fig3.jpg"> 图 3: 还原剂对 BMP2 完整性的影响。(A) BMP2-WT 暴露于抗坏血酸钠 (NaAsc) 与铜 (II) 硫酸盐 (丘索4) 的不同摩尔比率。通过 SDS 页和考马斯亮蓝染色 (图 A1) 和非还原条件 (图 A2) 对样品进行了分析。在减少的条件 (A1) 中, 红色箭头表示 BMP2-WT。在非还原条件 (A2) 中, 红色箭头表示 multimeric BMP2-WT。表示 BMP2 的条纹的带由蓝色箭头指示。图例: NCR -减少了 BMP2-WT 未经处理的情况;数控 BMP2-WT 未经处理;1:1 至 20:1-提高抗坏血酸钠的摩尔比值为丘索4。(B) BMP2-K3Plk 被耦合到 3-叠氮基-7-hydroxycoumarin, 使用 CuAAC 反应条件补充 THPTA。当耦合 3-叠氮基-7-hydroxycoumarin 成为一个荧光染料。图例: NC BMP2-WT 与 3-叠氮基-7-hydroxycoumarin 反应;7:1 至 20:1, THPTA 的摩尔比率增加到丘索4。<a href="https://cloudflare.jove.com/files/ftp_upload/56616/56616fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56616/56616fig4.jpg"> 图 4: 固定化 BMP2-K3Plk 的体外生物活性.固定化 BMP2-K3Plk 与德州红标记 BMPREC相互作用的代表性图片。转载 (改编), 并允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare.jove.com/files/ftp_upload/56616/56616fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56616/56616fig5.jpg"> 图 5: 固定化 BMP2-K3Plk 在基于细胞的检测中的生物活性。(A)碱性磷酸酶 (磷酸) 染色的代表性图片, 用珠子与 BMP2-K3Plk 功能性珠子结合处理。(B)碱性磷酸酶 (ALP) 染色治疗时可溶性 25 nM BMP2-K3Plk. 转载 (改编), 允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare.jove.com/files/ftp_upload/56616/56616fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry at JoVE.com

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

用骨形态发生蛋白 2 (BMP2) 掺杂的生物材料作为治疗非愈合性骨折的新方法。为了克服因无法控制的释放因素而产生的副作用, 我们提出了一个新的策略, 以现场直接固定的因素, 从而创造材料, 改善成骨能力。

点化学法现场定向固定骨形态发生蛋白2到固体表面

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

site-directed-immobilization-bone-morphogenetic-protein-2-to-solid

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Institutsteil Würzburg. 用骨形态发生蛋白 2 (BMP2) 掺杂的生物材料作为治疗非愈合性骨折的新方法。为了克服因无法控制的释放因素而产生的副作用, 我们提出了一个新的策略, 以现场直接固定的因素, 从而创造材料, 改善成骨能力。

视频: Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

荧光光谱法测定膜蛋白质折叠的热力学

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

监测与固态纳米孔蛋白质吸附

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

固定化酶和稳定的疏水盐改性的Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they&#39;re highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Q&#946;. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

Dibromomaleimide 二硫化学制备共轭诱导荧光 PEGylated 病毒样粒子

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...