OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱
Summary
在这里，我们提出了一个方案，通过酶形成蛋白质聚合物与受控序列结合蛋白质单体，并将其固定在表面进行单分子力光谱研究。
Abstract
近年来，化学和生物结合技术发展迅速，使蛋白质聚合物得以形成。然而，受控的蛋白质聚合过程始终是一个挑战。在这里，我们开发了一种酶方法，用于在合理控制的序列中逐步构建聚合蛋白。在这种方法中，蛋白质单体的C端是NGL用于蛋白质结合使用OaAEP1 （奥德兰尼亚亲基苯甲酰二甲苯肽酶） 1），而N端是一个可夹紧的TEV（烟草蚀刻病毒）裂解位点加L（ENLYFQ/GL）用于临时N端保护。因此，OaAEP1一次只能添加一个蛋白质单体，然后TEV蛋白酶在Q和G之间将N-终点板分刻，以暴露NH 2-Gly-Leu。然后，该装置已准备好进行下一次 OaAEP1 结扎。工程多蛋白通过展开单个蛋白质域使用原子力显微镜为基础的单分子力光谱（AFM-SMFS）进行检查。因此，本研究为多蛋白工程和固定化提供了一个有用的策略。
Introduction
与合成聚合物相比，天然多域蛋白具有统一的结构，具有控制良好的数量和子域1的类型。此功能通常导致改善生物功能和稳定性2，3。许多方法，如基于半胱氨酸的二硫化物结合和重组DNA技术，已经开发用于建立这种聚合蛋白与多个领域4，5，6，7。然而，前一种方法总是导致一个随机和不受控制的序列，而后者会导致其他问题，包括有毒和大型蛋白质的过度表达和复合蛋白与辅因子和其他微妙酶的纯化的困难。
为了迎接这一挑战，我们开发了一种酶法，利用蛋白胶合OaAEP1与蛋白酶TEV8、9相结合，将蛋白质单体结合在一起，以循序渐进的方式用于聚合物/多蛋白。OaAEP1是一种严格而高效的内肽酶。如果N-终点为Gly-Leu残留物（GL），则两种蛋白质可于30分钟内通过OaAEP1通过两个终点线（NGL）进行共价连接，而C-终点为NGL残留物10。然而，使用OaAEP1只将蛋白质单体与蛋白质聚合物联系起来，其序列与基于半胱氨酸的耦合方法一样不受控制。因此，我们设计了具有可移动TEV蛋白酶位点的蛋白质单元的N-终点，以及作为ENLYFQ/G-L-POI的亮氨酸残留物。在 TEV 裂解之前，N 终端不会参与 OaAEP1 连接。然后，N-总站的GL残留物，与进一步的OaAEP1结扎兼容，在TEV裂解后暴露。因此，我们实现了多蛋白的连续酶生物合成方法，序列控制相对较好。
在这里，我们的分步酶合成方法可用于多蛋白样品制备，包括序列控制和不受控制，以及蛋白质固定用于单分子研究，特别是对于复杂系统，如金属蛋白。
此外，基于AFM的SMFS实验使我们能够确认蛋白质聚合物在单分子水平上的结构和稳定性。单分子力光谱，包括AFM，光学钳子和磁钳，是纳米技术中一般工具，以机械方式操纵生物分子，测量其稳定性11，12，13，14，15，16，17，18，19，20。单分子AFM已广泛应用于蛋白质（非）折叠21，22，23，24，25，受体-配体相互作用的强度测量26，27，28，29，30，31，32，33，34， 35，无机化学键20，36，37，38，39，40，41，42，43和金属配体键在金属蛋白44，45，46，47，48，49，50.在这里，使用单分子AFM验证基于相应蛋白质展开信号的合成多蛋白序列。
Protocol
1. 蛋白质生产 基因克隆 购买感兴趣的蛋白质（POI）的基因编码：泛素、鲁布雷多辛（RD）51、纤维素结合模块（CBM）、Dockerin-X域（XDoc）和从鲁米诺球菌浮躁、烟草蚀刻病毒（TEV）蛋白酶、弹性蛋白样多肽（ELPs）的内聚力。 进行聚合酶链反应，并使用三限消化酶系统BamHI-BglII-KpnI对不同蛋白质片段的基因进行重组。<…
Representative Results
OaAEP1结扎在相邻蛋白质之间引入的NGL残留物不会影响聚合物中的蛋白质单体稳定性，因为展开力（<Fu>）和轮廓长度增量（<+Lc>;）与上一项研究（图1）相当。红霉素蛋白的纯化结果如图2所示。为了证明TEV裂解后的蛋白质与以下OaAEP1结扎相容，以构建具有控制序列的蛋白质聚合物，结构高效，图3提供了SDS-PAGE图像作…
Discussion
我们描述了一种酶生物合成和多蛋白固定化方案，并验证了基于AFM的SMFS的多蛋白设计。这种方法提供了一种新方法，以设计的顺序构建蛋白质聚合物，补充了以前多蛋白工程和固定4、6、52、53、54、55、56、57、58、59、60、61的方法。</su…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（授权号21771103，21977047），江苏省自然科学基金（授权号21771103）的支持。BK20160639）和江苏省双川计划。
Materials
| iron (III) chloride hexahydrate | Energy chemical | 99% | |
| Zinc chloride | Alfa Aesar | 100.00% | |
| calcium chloride hydrate | Alfa Aesar | 99.9965% crystalline aggregate | |
| L-Ascorbic Acid | Sigma Life Science | Bio Xtra, ≥99.0%, crystalline | |
| (3-Aminopropyl) triethoxysilane | Sigma-Aldrich | ≥99% | |
| sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate | Thermo Scientific | 90% | |
| Glycerol | Macklin | 99% | |
| 5,5'-dithiobis(2-nitrobenzoic acid) | Alfa Aesar | ||
| Genes | Genscript | ||
| Equipment | |||
| Nanowizard 4 AFM | JPK Germany | ||
| MLCT cantilever | Bruker Corp | ||
| Mono Q 5/50 GL | GE Healthcare | ||
| AKTA FPLC system | GE Healthcare | ||
| Glass coverslip | Sail Brand | ||
| Nanodrop 2000 | Thermo Scientific | ||
| Avanti JXN-30 Centrifuge | Beckman Coulter | ||
| Gel Image System | Tanon |
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Diesen Artikel zitieren
Deng, Y., Zheng, B., Liu, Y., Shi, S., Nie, J., Wu, T., Zheng, P. OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy. J. Vis. Exp. (156), e60774, doi:10.3791/60774 (2020).