J.J.G. 承认美国德州科学基金会 (DMR-1654405) 和德克萨斯癌症预防研究所 (CPRIT) (RP170752s) 的支持。

1. 准备
<ol><li>制作溶源性汤 (磅) 琼脂和倒盘12。</li>
	<li>
用含有 wtQβ涂层蛋白序列的 pET28 质粒转化 BL21 (DE3)。
	<ol>
<li>解冻大肠杆菌BL21 (DE3) 在冰浴中的主管细胞。在离心管中放置50个细胞的 ul。</li>
		<li>将质粒的 2 ul 添加到一管中, 轻轻地轻拂管子。然后在冰上孵育30分钟。</li>
		<li>热休克细胞四十五年代在水浴那是在确切地42°c。热震后立即将管子放回冰浴中, 孵化5分钟。</li>
		<li>添加950不含任何抗生素的 LB 介质的 ul。</li>
		<li>在37摄氏度的60分钟内, 以 200 rpm 的文化摇摆。</li>
		<li>板材 100 ul 的文化在 LB 琼脂板 (与卡那霉素) 和孵化的板块隔夜在37摄氏度。需要时选择白色菌落。</li>
	</ol>
</li>
	<li>
制作超级最佳的汤 (呜咽) 介质。
	<ol>
<li>蒸压釜两个2升困惑锥形烧瓶 superdry 循环。</li>
		<li>在无菌环境中, 称量并添加20.0 克胰蛋白胨, 5.0 克酵母萃取物, 2.469 克无水硫酸镁, 0.584 克氯化钠和0.186 克氯化钾到每瓶。</li>
		<li>把体积在每瓶和高压釜的液体循环中的超纯水1升。</li>
		<li>一旦呜咽的媒体已经达到室温后热处理, 添加1毫升卡那霉素 (100 毫克/毫升) 每公升的媒体和存储在4摄氏度。</li>
	</ol>
</li>
	<li>
使0.1 米磷酸钾缓冲液 (pH 7.00)。
	<ol>
<li>在500毫升的超纯水中溶解68.045 克磷酸钙钾, 使1米磷酸钙溶液。</li>
		<li>在500毫升超纯水中溶解87.09 克钾二元, 使二元钾溶液1米。</li>
		<li>将38.5 毫升的钾磷酸钙溶液和61.5 毫升的二元钾添加到1升瓶中。</li>
		<li>如果需要, 将 pH 值调整为 7.00, 并使其体积为1升。</li>
	</ol>
</li>
	<li>
在0.1 米磷酸钾缓冲液中5–40% 蔗糖梯度 (pH 值为 7.00)。
	<ol>
<li>在50毫升离心管中, 用 5–40% (增加 5%) 蔗糖溶解于0.1 米磷酸钾缓冲液 (pH 7.00) 制备溶液。</li>
		<li>用一根长针注射器在38毫升的聚碳酸酯管底部沉积3.3 毫升5% 蔗糖溶液, 并对五其他管进行重复。</li>
		<li>小心地将3.3 毫升的10% 蔗糖溶液放在管子的底部, 小心地除去针, 以免扰乱渐变。重复其他五管。 
		 </li>
	</ol>
</li>
	<li>继续存入3.3 毫升的蔗糖溶液层, 每管增加15% 到 40%, 同时小心不干扰梯度。</li>
	<li>完成后, 用铝箔覆盖渐变的顶部, 并存储在-80 摄氏度。</li>
</ol>2. Qβ的表达
<ol><li>用1:1 漂白剂/乙醇擦拭工作台面积。</li>
	<li>在无菌环境中, 通过将大肠杆菌BL21 (DE3) 的单个菌落添加到3毫升的无菌环境中, 使两个3毫升的起始培养物处于不细菌的氛围中。</li>
	<li>在一个 250 rpm 在37摄氏度和0% 相对湿度 (rH) 房间一夜之间增长。</li>
	<li>
在呜咽媒体中接种起始文化:
	<ol>
<li>从振动筛中取走3毫升的起始培养物, 在无菌环境中, 将每种起始文化倒入两个 2 l 困惑的锥形烧瓶中的一个, 每瓶有1升的新鲜呜咽介质。</li>
		<li>将接种的介质放在 250 rpm 的振动筛上, 在37摄氏度和0% 个 rH 室。</li>
	</ol>
</li>
	<li>在37摄氏度和 0% rH 室中, 在 250 rpm 的振动筛上种植细菌, 直到 OD600到达0.9–1.0。</li>
	<li>用 P1000 吸管添加1毫升1米异丙基β-d-1 thiogalactopyranoside (IPTG), 诱导蛋白表达。</li>
	<li>在37摄氏度和 0% rH 房间过夜时, 将媒体放在 250 rpm 的振动筛上。</li>
	<li>第二天早上从振动筛中取出介质, 用1000毫升的瓶子在 20621 x 克上用1小时在4摄氏度处收割细胞。</li>
	<li>
丢弃上清液并收集细胞颗粒。
	<ol>
<li>将上清液倒入一瓶约5毫升漂白剂, 以杀灭细菌。这是浪费。</li>
		<li>使用铲刮从离心瓶底部的细胞颗粒, 并把球团成50毫升离心管。</li>
	</ol>
</li>
</ol>3. Qβ的纯化
<ol><li>并用重悬20–30毫升0.1 米磷酸钾缓冲细胞颗粒 (pH 7.00)。</li>
	<li>请确保泥沙没有块, 并根据制造商的协议溶解单元格使用 microfluidizer 处理器 (请参见材料表)。溶解细胞至少两次, 以增加粒子的产量。</li>
	<li>离心250毫升离心机瓶在 20621 x g 1 小时在4摄氏度的裂解。</li>
	<li>丢弃颗粒, 测量 mL 中清液的体积。将该值乘以 0.265, 并将硫酸铵的 g 量加到上清。</li>
	<li>在 200 rpm 的搅拌板上搅动4摄氏度, 至少1小时, 以沉淀出蛋白质。</li>
	<li>离心机在250毫升瓶在 20621 x g 1 小时在4°c。</li>
	<li>用大约10毫升的0.1 米磷酸钾缓冲液 (pH 7.00) 丢弃上清和并用重悬颗粒。</li>
	<li>将等量的1:1 氯仿/正丁醇添加到粗样品中, 用涡流混合几秒钟。</li>
	<li>离心机在38毫升管在 20621 x g 30 分钟在4°c。</li>
	<li>使用巴斯德吸管回收顶部水层。小心不要采取在水和有机层之间形成的凝胶状层。</li>
	<li>解冻六5–40% 预先做的蔗糖梯度和装载大约2毫升提取物到每个。</li>
	<li>Ultracentrifuge 在 99582 x g 为16小时在4°c 与自由减速。</li>
	<li>发光二极管 (LED) 光在每个管下, 蓝带应该变得可见。用长针注射器回收这些微粒。</li>
	<li>Ultrapellet 的粒子在 370541 x g 2.5 小时在4摄氏度。</li>
	<li>用0.1 米磷酸钾缓冲液 (pH 为 7.00), 丢弃上清和并用重悬纯化颗粒的透明颗粒。</li>
</ol>4. 产品的数量和确认
<ol><li>使用布拉德福德检测来量化产品13。</li>
	<li>运行还原和不还原的月桂酸钠聚丙烯酰胺凝胶电泳 (SDS 页) 确认产品14。 注: 减少 SDS 页用于确定涂层蛋白的分子量;不缩减的 SDS 页面是确认高阶结构。</li>
</ol>5. Qβ上的共轭 DB 化合物
<ol><li>
减少 Qβ上的二硫化物。
	<ol>
<li>将0.0020 克的三 (2-羧乙基) 膦 (TCEP) 溶解成1毫升的超纯水, 以制造100x 的库存溶液。 注: 在还原前准备新鲜 TCEP。</li>
		<li>添加200µL 的 Qβ (5 毫克/毫升) 到离心管。</li>
		<li>通过添加20µL 100x TCEP 库存解决方案进行跟踪。</li>
		<li>室温孵育1小时。</li>
	</ol>
</li>
	<li>
用 dibromomaleimide 聚乙二醇 (DB PEG) 重新桥降二硫醚。
	<ol>
<li>在100µL 的 DMF 中溶解0.0017 克 dibromomaleimide 聚乙二醇 (DB PEG)。</li>
		<li>添加680µL 10 毫米磷酸磷酸钠溶液 (pH 5.00)。</li>
		<li>将减少的 Qβ加入到 DB 的溶液中, 观察 365 nm 紫外线灯下的混合过程。明亮的黄色荧光可以立即可视与365纳米手持紫外线灯在混合。</li>
		<li>让反应在室温下 (RT) 在烤上一夜之间进行。</li>
	</ol>
</li>
	<li>用离心过滤器 (COMW = 10 kDa) 净化反应混合物三次使用 1x PBS 在 3283 x g 20 分钟在4°c。</li>
	<li>通过非还原 SDS 页和本机琼脂糖凝胶电泳对共轭进行监测。 注: VLPs 在本机琼脂糖凝胶中作为完整的颗粒运行, 并根据其电荷、大小和形状分离。</li>
</ol>

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作者声明他们没有竞争的财政利益。

与较小的蛋白质纯化相比, 纯化噬菌体 Qβ的一个独特步骤是蔗糖梯度离心。在氯仿/正丁醇萃取步骤后, 用5-40% 蔗糖梯度进一步纯化 Qβ。在离心过程中, 粒子根据它们的大小分开。更大的微粒旅行到更高的密度区域, 而小微粒停留在低密度区域。Qβ移动到梯度的下第三, 并保持在那里, 而较小的蛋白质杂质被困在离心管的顶部。Qβ的胶体悬浮在蔗糖梯度中表现出强烈的廷德尔散射, 当 LED 光从下面照射...

使用纳米病毒衣壳已成为一个令人兴奋的领域, 目的是扩大在生物医学研究领域的应用范围 1, 2, 3.Recombinantly 表达的病毒样粒子 (VLPs) 的结构来源于病毒, 但它们缺乏原始的病毒遗传物质, 使得它们无传染性的蛋白质纳米粒子。由于表面特征是基因编制的, 并且每个衣壳在它之前和之后被表达与它是相同的, 有可能知道氨基酸的无功侧链的位置和数量以原子精确度。在许多情况下, 外部和内部表面都具有多种溶剂暴露的氨基酸残留物, 这可以通过 bioconjugation 反应进行功能化--在分子和合成物之间形成共价键的反应。分子4,5。
Bioconjugation 反应帮助生物分子在相对简单的方式有更多不同的功能。感兴趣的分子, 如治疗药物6, 荧光标记7和聚合物8,9可以预先合成和特征, 然后它们被附着在 VLPs 表面上。在生物医学和生物材料研究中, 一个特别常见的 vip 是基于噬菌体 Qβ的 vip, 正如 recombinantly 所表达的, 它是一个28纳米 icosahedral 病毒衣壳10。Qβ上最常见的反应部位是 lysines, 尽管最近我们已经将 dibromomaleimide 化合物的成功共轭11与二硫化物, 通过 Qβ-贝克反应将 Haddleton 的孔隙线进行了传递。反应的收益率很好, 同样重要的是, 不会失去粒子的热稳定性。同时, 这种反应产生共轭诱导荧光, 可用于跟踪这些微粒对细胞的吸收。在这项工作中, 我们展示了聚乙二醇 (PEG) 在 Qβ表面上的共轭, 通过 Haddleton 反应, 这导致了一个明亮的黄色荧光。这些粒子可以被跟踪, 因为它们被细胞所接受。本协议将帮助研究人员生成基于 Qβ的新型荧光 PEGylated 蛋白质纳米粒子, 尽管它的原理适用于含有溶剂暴露二硫化物的许多其它 VLPs 中的一种。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LB Broth (Miller)&#160;</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">1.10285.0500</td>
			<td style="width:167px;"></td>
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			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">T60060-1000.0</td>
			<td></td>
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		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Yeast Extract, Poweder</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">Y20020-1000.0</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anhydrous magnesium sulfate</td>
			<td style="width:71px;">P212121</td>
			<td style="width:119px;">CI-06808-1KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride (Crystalline/Certified ACS)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Chloride</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP366-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Elga PURELAB Flex 3 Water Purification System</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">4474524</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP362-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Dibasic Anhydrous</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">P288-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">S25590B</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP2818500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">I6758-1G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fiberlite F10-4x1000 LEX rotor&#160;</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">096-041053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium Sulfate</td>
			<td style="width:71px;">P212121</td>
			<td style="width:119px;">KW-0066-5KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">32614-M6</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-Butanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">A399-4</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">SW 28 Ti Rotor, Swinging Bucket, Aluminum</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">342204: SW 28 Ti Rotor/ 342217: Bucket Set</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Type 70 Ti Rotor, Fixed Angle, Titanium, 8 x 39 mL,</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">337922</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">PI23200</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">TRIS Hydrochloride</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">T60050-1000.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tetramethylethylenediamine</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">J63734-AC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">C4706-2G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2 3-Dibromomaleimide 97%</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">553603-5G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polythylene Glycol</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">41561-22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Phosphate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">AC424375000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acrylamide/bis-Acrylamide</td>
			<td style="width:71px;">P212121</td>
			<td style="width:119px;">RP-A11310-500.0</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium dodecyl sulfate</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium Persulfate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP179-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FV3000 confocal laser scanning microscope</td>
			<td style="width:71px;">Olympus&#160;</td>
			<td>FV3000&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Labnet Revolver Adjustable Rotator&#160;</td>
			<td style="width:71px;">Thomas Scientific&#160;</td>
			<td>1190P25&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1000 mL Sorvall High Performance Bottle, PC, with Aluminum Cap&#160;</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">010-1459</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nalgene Centrifuge Bottles with Caps, Polypropylene Copolymer</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">3141-0250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc Round-bottom tubes; 38 mL; PC</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">3117-0380</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2 L Narrow Mouth Erlenmeyer Flasks with Heavy Duty Rim</td>
			<td style="width:71px;">Pyrex</td>
			<td style="width:119px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra-4 Centrifugal Filter Units</td>
			<td style="width:71px;">Millipore Sigma</td>
			<td style="width:119px;">UFC801024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">M-110P Microfluidizer Materials Processor</td>
			<td style="width:71px;">Microfluidics</td>
			<td style="width:119px;">M-110P</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">3117-0380PK</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bottle, with Cap Assembly, Polycarbonate</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">41121703</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cylinder, Graduated - Polypropylene 250 mL</td>
			<td style="width:71px;">PolyLab</td>
			<td style="width:119px;">80005</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">533LS-E Series Steam Sterilizers</td>
			<td style="width:71px;">Getinge</td>
			<td style="width:119px;">533LS-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TrueLine, Cell Culture Plate, Treated, PS, 96 Well, with Lid</td>
			<td style="width:71px;">LabSource</td>
			<td style="width:119px;">D36-313-CS</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon 15 mL Conical Centrifuge Tube</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microcentifuge Tube: 1.5mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">VWR Os-500 Orbital Shaker</td>
			<td style="width:71px;">VWR Scientifc Products</td>
			<td style="width:119px;">14005-830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetra Handcast systems</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;">1658000FC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polypropylene, 250 mL</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">41121703</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Spectrofluorometer NanoDrop</td>
			<td style="width:71px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">3300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Long Needle&#160;</td>
			<td style="width:71px;">Hamilton&#160;</td>
			<td style="width:119px;">7693</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Exel International 5 to 6 cc Syringes Luer Lock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-841-46</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P200 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P100 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P20 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F123600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P10 Pipetman</td>
			<td style="width:71px;">Gilson</td>
			<td style="width:119px;">F144802</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Intel Weighing PM-100 Laboratory Classic High Precision Laboratory Balance</td>
			<td style="width:71px;">Intelligent Weighting Technology</td>
			<td style="width:119px;">IWT_PM100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon 50 mL Conical Centrifuge Tube</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4&#8211;15% Mini-PROTEAN TGX Gel, 10 well, 50 &#181;l</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;">456-1084</td>
			<td></td>
		</tr>
	</tbody>
</table>

making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry

最近在生物医学和材料研究中出现的病毒样粒子 (VLPs) 的增加, 可以归因于它们的生物合成、离散大小、遗传可编程性和生物降解能力的简化。虽然他们很容易 bioconjugation 反应, 添加合成配体在其表面上, bioconjugation 方法的范围, 这些水中出生的衣壳是相对有限的。为了促进功能性生物材料研究的方向, 必须考虑非传统的 bioconjugation 反应。本协议中描述的反应使用 dibromomaleimides 在噬菌体 Qβ的基础上引入了一个 vip 的溶剂暴露二硫化物键的新功能。此外, 最终产品是荧光, 它具有生成追踪体外探针使用商业上可用的筛选器集的额外好处。

dibromomaleimide 衍生物可通过 dibromomaleimide 酸酐与主胺15之间的缩合反应合成。另外, 使用 n-羧酸激活 34-dibromomaleimide 的温和合成方法16在这里被开发了与 methoxypolyethylene 乙二醇 (peg) 反应产生 DB peg (图 1)。核磁共振用于识别复合结构 (图 2)。Qβ vip 是一个28纳米 icosahedral 蛋白质纳米粒子, 这是由180?...

Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

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

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

在这里, 我们提出一个程序, 荧光 functionalize 二硫化物 Qβ vip 与 dibromomaleimide。我们描述了 Qβ的表达和纯化, dibromomaleimide 功能分子的合成, dibromomaleimide 和 Qβ的共轭反应。生成的黄色荧光共轭粒子可作为细胞内的荧光探针。

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

making-conjugation-induced-fluorescent-pegylated-virus-like-particles

Research

JoVE Journal

Bioengineering

6.8K 次观看.  University of Texas at Dallas. 在这里, 我们提出一个程序, 荧光 functionalize 二硫化物 Qβ vip 与 dibromomaleimide。我们描述了 Qβ的表达和纯化, dibromomaleimide 功能分子的合成, dibromomaleimide 和 Qβ的共轭反应。生成的黄色荧光共轭粒子可作为细胞内的荧光探针。

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

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 ...

科研

生物工程

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

可生物降解聚合物颗粒的淋巴内结注射

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

通过通过纳米孔可调纳米易位流速Zeta电位测定:利用DNA修饰颗粒为例

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

Nanoplasmonic 光学晶格中微粒子的俘获

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

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

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

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 ...

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 ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.