蛋白质生物缀合物的合成<em>通过</em>半胱氨酸马来酰亚胺化学
Summary
该协议细节对于含有蛋白质马来酰亚胺，包括试剂纯化，反应条件，生物共轭纯化和生物共轭表征半胱氨酸的生物结合所需的重要步骤。
Abstract
The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine – maleimide coupling of proteins to other proteins, dyes, drugs or polymers.
Introduction
生物缀涉及共价连接一种生物分子与另一个或同一个合成的分子，例如染料，药物或聚合物。蛋白质的生物耦合方法现在广泛应用于许多化学，生物和纳米技术研究小组使用，应用范围从荧光染料标记1,2，使蛋白质（抗体）-prodrugs 3（抗体偶联药物- ADC）的蛋白二聚体4,5的合成通过在纳米8和系统化学9中使用的自组装蛋白-聚合物杂种6,7。
用于生物结合化学的特异性，而并非总是关键的，是对大多数功能性蛋白质生物共轭物极为重要，以便不与目标蛋白的活性位点干扰。提供了理想生物缀反应需要满足若干标准，包括：感兴趣的蛋白质上ⅰ）靶向稀有或独特的位点，ⅱ）是有选择性的实现这一目标，ⅲ）非变性条件下进行，以避免蛋白质折叠和iv）是高产作为目标蛋白质通常只适用于子毫摩尔浓度。马来酰亚胺-半胱氨酸迈克尔加成接近满足所有这些标准，并具有该原因长权利生物共轭化学10的字段的特殊状态。这是因为，ⅰ）在其表面上仅含有一个半胱氨酸残基的许多蛋白质可以遗传工程那里，ⅱ）在正确的pH值的反应是朝着半胱氨酸高度选择性，ⅲ）它可顺利地进行在水性缓冲液和iv）它是非常快的与马来酰亚胺的二阶速率常数报告超过5000 M -1秒-1在某些情况下11含有半胱氨酸的蛋白质。提供感兴趣的蛋白质可以容忍有机的一个小的（≈5-10％）量的共溶剂12，几乎任何马来酰亚胺官能化的染料，婆lymer，表面或另一种蛋白可以链接到蛋白质。此外，马来酰亚胺是对蛋白质比碘乙酰胺，这是更容易在升高的pH值的其他亲核体进行反应的半胱氨酸更具体;比这需要在酸性pH值被保持，以防止二硫键交换13基于二硫键缀合更稳定。
此处我们报告马来酰亚胺官能化的分子的缀合的通用协议包含使用钌（Ⅱ）基的发色团和氧化还原蛋白细胞色素C作为例子之间的反应的单个半胱氨酸残基的蛋白质。这个协议同样适用于含有可接近表面的半胱氨酸残基和相应的马来酰亚胺官能化的靶大多数其他蛋白，无论是另一种蛋白，荧光染料，发色团或合成聚合物。
Protocol
Representative Results
Discussion
一个生物结合前的原料纯化是非常重要的。从商业的重组来源得到的蛋白质常含有目的蛋白质，其可具有不同的表面化学和反应的其它同种型。例如，在所描述的生物结合，市售细胞色素C包含两个异1和异2色素C 12,14,17的混合物。异2和异1形式的细胞色素c在很大程度上是同源的，与主要的区别是异1细胞色素C的C-末端附近的一个游离半胱氨酸残基的存在?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank the Australian Research Council (ARC) for ARC Future Fellowship (FT120100101) and ARC Centre of Excellence CE140100036) grants to P.T. and the Mark Wainwright Analytical Centre at UNSW for access to mass spectrometry and NMR facilities.
Materials
| sodium dihydrogen phosphate | Sigma-Aldrich | 71496 | |
| sodium hydroxide | Sigma-Aldrich | 71691 | |
| sodium chloride | Sigma-Aldrich | 73575 | |
| cytochrome c, from saccaromyces cerevisiae | Sigma-Aldrich | C2436 | |
| dithiothreitol | Sigma-Aldrich | 43819 | |
| TSKgel SP-5PW | Sigma-Aldrich | Tosoh SP-5PW, 07161 | 3.3 mL strong cation exchange column |
| Amicon Ultra-15 | Merck-Millipore | UFC900308 | 3.5 kDa spin filter |
| Slide-A-Lyzer mini dialysis units | Thermo Scientific | 66333 | 3.5 kDa dialysis cassetes |
| Ru(II) bisterpyridine maleimide | Lab made | see ref (14) | |
| acetonitrile | Sigma-Aldrich | A3396 | |
| ethylenediaminetetraacetic acid | Sigma-Aldrich | 03609 | |
| tris(2-carboxyethyl)phosphine hydrochloride | Sigma-Aldrich | 93284 | |
| imidazole | Sigma-Aldrich | 56749 | |
| nickel acetate | Sigma-Aldrich | 244066 | |
| AcroSep IMAC Hypercell column | Pall | via VWR: 569-1008 | 1 mL IMAC column |
| 0.2 micron cellulose membrane filter | Whatman | Z697958 | 47 mm filter for buffers |
| 0.2 micron PVDF membrane filter | Merck-Millipore | SLGV013SL | syringe filters for proteins |
| hydrochloric acid | Sigma-Aldrich | 84426 | extremely corrosive! Use caution |
| caffeic acid | Sigma-Aldrich | 60018 | MALDI matrix |
| trifluoroacetic acid | Sigma-Aldrich | 91707 | extremely corrosive! Use caution |
| SimplyBlue SafeStain | Thermo Scientific | LC6060 | Coomassie blue solution |
| NuPAGE Novex 12% Bis-Tris Gel | Thermo Scientific | NP0342BOX | precast protein gels |
| SeeBlue Plus2 Pre-stained Protein Standard | Thermo Scientific | LC5925 | premade protein ladder |
| NuPAGE LDS Sample Buffer (4X) | Thermo Scientific | NP0008 | premade gel sample buffer |
| NuPAGE Sample Reducing Agent (10X) | Thermo Scientific | NP0004 | premade gel reducing agent |
| NuPAGE MES SDS Running Buffer (20X) | Thermo Scientific | NP0002 | premade gel running buffer |
| Voyager DE STR MALDI reflectron TOF MS | Applied Biosystems | ||
| Acta FPLC | GE | Fast Protein Liquid Chromatography | |
| Cary 50 Bio Spectrophotometer | Varian-Agilent | UV-Vis | |
| Milli-Q ultrapure water dispenser | Merck-Millipore | ultrapure water | |
| Low volume UV-Vis Cuvette | Hellma | 105-201-15-40 | 100 microliter cuvette |
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