使用生物素标签研究蛋白质-核酸相互作用的体外生化分析
1State Key Laboratory of Reproductive Medicine,Nanjing Medical University, 2The Affiliated Hospital of Hangzhou Normal University, 3School of Life Science and Technology,ShanghaiTech University, 4Department of Tissue and Embryology, School of Basic Medical Sciences, Hubei Provincial Key Laboratory of Developmentally Originated Disease,Wuhan University
Summary
这里介绍的是使用生物素标签的体外生化测定方案,可广泛用于研究蛋白质-核酸相互作用。
Abstract
蛋白质-核酸相互作用在生物过程(如转录、重组和RNA代谢)中起着重要作用。研究蛋白质-核酸相互作用的实验方法需要使用荧光标记、放射性同位素或其他标签来检测和分析特定的目标分子。Biotin 是一种非放射性核酸标签,通常用于电泳移动转移测定 (EMSA),但一直没有定期用于监测核酸过程中的蛋白质活性。该协议说明了生物锡标签在体外酶反应过程中的效用,证明该标签与一系列不同的生化测定有效。具体来说,根据之前使用放射性同位素32P标记基质的发现,通过生物素标记EMSA确认MEIOB(一种专门参与美智重组的蛋白质)是一种DNA结合蛋白,即MOV10(一种RNA合壳)可解析生物素标记的RNA双工结构,而MEIOB可使生物素标记的单链DNA。研究表明,生物素能够在体外各种核酸相关生化检测中替代32P。
Introduction
蛋白质-核酸相互作用涉及许多重要的细胞过程,如DNA修复、复制、转录、RNA处理和翻译。蛋白质与染色质内特定DNA序列的相互作用是严格控制转录级别1的基因表达所必需的。对大量编码和非编码RNA的精确转录后调节需要任何蛋白质和RNA2之间广泛而复杂的相互作用。这些基因表达调控机制层包括一系列动态的分子间事件,通过转录/表观遗传因子或RNA结合蛋白与其核酸靶点的相互作用以及核酸靶点进行协调。蛋白质-蛋白质相互作用。为了解剖体内的蛋白质是否与核酸直接或间接相关,以及这种关联是如何发生和达到高潮的,进行体外生化测定,以检查相关蛋白质的结合亲和力或酶活性。设计DNA和/或RNA的基质。
已经开发了许多技术来检测和表征核酸-蛋白质复合物,包括电泳移动移位测定(EMSA),也称为凝胶缓动测定或带移检测3、4、5.EMSA 是一种多功能、灵敏的生化方法,广泛用于研究蛋白质与核酸的直接结合。EMSA依靠凝胶电泳带的变化,通常使用化学发光来检测生物素标签6,7,荧光素标签8,9的荧光,或通过放射性32P标签的自动辐射标签10,11。生化研究的其他用途是鉴定和表征蛋白质的核酸处理活性,如核酸反应,以评估核酸基质的裂解产物12,13,14和DNA/RNA结构展开测定,以评估赫利箱活性15,16,17。
在这种酶活性测定中,放射性同位素标记或荧光素标记的核酸由于其高灵敏度,经常被用作基质。对涉及32个P标记放射性追踪器的酶反应的放射图分析发现是敏感和可重复的18。然而,在世界上越来越多的实验室中,放射性同位素的使用受到限制甚至被禁止,因为与潜在接触相关的健康风险。除了生物安全问题外,其他缺点是使用放射性同位素工作所需的设备、所需的放射性许可证、32 P 的短半寿命(约 14天),以及探测器质量因无线电辐射。因此,开发了替代非同位素方法(即用荧光光对探针标记探头,通过荧光成像19进行检测)。但是,使用荧光标记的探头时,需要高分辨率成像系统。生物素是一种常用的标签,很容易与蛋白质和核酸等生物大分子结合。生物链球菌系统高效运行,提高了检测灵敏度,同时不增加非特异性背景20,21。除EMSA外,生物锡还广泛用于蛋白质纯化和RNA下拉,包括22、23、24。
该协议成功地使用生物素标记的核酸作为包括EMSA的体外生化检测的基质,此外还有未普遍使用的酶反应。MEIOB OB域被构造,两个突变体(截流和点突变)表示为GST融合蛋白25,26,27,以及小鼠MOV10重组FLAG融合蛋白16。本报告强调了这种用于蛋白质纯化和生物锡标记检测的合并方案的有效性,用于其他实验目的。
Protocol
1. 蛋白质制备 MEIOB 和 MOV10 表达式构造生成 cDNA 表达式构造编码小鼠 MEIOB-A、C 和 E (图 1A) 和 MOV10。 设置每个片段的聚合酶链反应 (PCR) 反应。在最终组合 1 μL 小鼠 cDNA(来自 C57BL/6 小鼠睾注)、1 μL dNTP、2 μL 10 μM 正向底漆、2 μL 10 μM 反向底漆、1 μL DNA 聚合酶、25 μL 2x PCR 缓冲液和 18 μL 双蒸馏 H2O (ddH2O)体积为 50 μL。注:…
Representative Results
MEIOB的蛋白质结构和本研究中使用的表达结构如图1A所示。 MEIOB 中的 OB 折叠是紧凑的桶状结构,可以识别和与单链核酸相互作用。其中一个OB域(aa 136-307,构造A)结合单绞合DNA(sSDNA),截断蛋白(aa 136-178截断,构造C)和点突变形式(R235A突变,构造E)的MEIOB没有DNA结合活性26。GST-MEIOB融合蛋白在BL21细菌中被过度表达,随后的分离步骤导致通过Coomassie蓝色染色和?…
Discussion
研究蛋白质-核酸相互作用对于我们理解不同生物过程背后的分子机制至关重要。例如,MEIOB是哺乳动物25、26、27中梅氏症和生育力所必需的睾度特异性蛋白质。MEIOB包含一个OB域,它与单链DNA结合,并表现出3’至5’外源酶活性26,这与其在美质重组过程中的生理相关性直接相关。作为另一个例子,MOV10是一种具有无处不?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢P.Jeremy Wang(宾夕法尼亚大学)所做的有益的编辑和讨论。我们还感谢西格丽德·埃卡德特的语言编辑。K. Z. 获得中国国家重点研发项目(2016YFA0500902,2018YFC1003500)和国家自然科学基金(31771653)的支持。L. Y. 得到了国家自然科学基金(81471502,31871503)和江苏省创新创业项目的支持。J.N.得到了浙江省医学科技项目(2019KY499)的支持。M.L.得到了国家自然科学基金(31771588)和”千名青年人才计划”的资助。
Materials
| Equipment | |||
| Centrifuge | Eppendorf, Germany | 5242R | |
| Chemiluminescent Imaging System | Tanon, China | 5200 | |
| Digital sonifer | Branson, USA | BBV12081048A | 450 Watts; 50/60 HZ |
| Semi-dry electrophoretic blotter | Hoefer, USA | TE77XP | |
| Tube Revolver | Crystal, USA | 3406051 | |
| UV-light cross-linker | UVP, USA | CL-1000 | |
| Materials | |||
| Amicon Ultra-4 Centrifugal Filter | Milipore, USA | UFC801096 | 4 ml/10 K |
| Nylon membrane | Thermo Scientific, USA | TG263940A | |
| TC-treated Culture Dish | Corning, USA | 430167 | 100 mm |
| TC-treated Culture Dish | Corning, USA | 430597 | 150 mm |
| Microtubes tubes | AXYGEN, USA | MCT-150-C | 1.5 mL |
| Tubes | Corning, USA | 430791 | 15 mL |
| Reagents | |||
| Ampicillin | SunShine Bio, China | 8h288h28 | |
| Anti-FLAG M2 magnetic beads | Sigma, USA | M8823 | |
| ATP | Thermo Scientific, USA | 591136 | |
| BCIP/NBT Alkaline Phosphatase Color Development Kit | Beyotime, China | C3206 | |
| CelLyticTM M Cell Lysis Reagent | Sigma, USA | 107M4071V | |
| ClonExpress II one step cloning kit | Vazyme, China | C112 | |
| Chemiluminescent Nucleic Acid Detection Kit | Thermo Scientific, USA | T1269950 | |
| dNTP | Sigma-Aldrich, USA | DNTP100-1KT | |
| DMEM | Gibco, USA | 10569044 | |
| DPBS buffer | Gibco, USA | 14190-136 | |
| EDTA | Invitrogen, USA | AM9260G | 0.5 M |
| EDTA free protease inhibitor cocktail | Roche, USA | 04693132001 | |
| EndoFree Maxi Plasmid Kit | Vazyme, China | DC202 |
|
| FastPure Gel DNA Extraction Mini Kit | Vazyme, China | DC301-01 | |
| FBS | Gibco, USA | 10437028 | |
| FLAG peptide | Sigma, USA | F4799 | |
| Glycerol | Sigma, USA | SHBK3676 | |
| GST Bulk Kit | GE Healthcare, USA | 27-4570-01 | |
| HEPES buffer | Sigma, USA | SLBZ2837 | 1 M |
| IPTG | Thermo Scientific, USA | 34060 | |
| KoAc | Sangon Biotech, China | 127-08-02 | |
| Lipofectamin 3000 Transfection Reagent | Thermo Scientific, USA | L3000001 | |
| MgCl2 | Invitrogen, USA | AM9530G | 1 M |
| NaCl | Invitrogen, USA | AM9759 |
5 M |
| NP-40 | Amresco, USA | M158-500ML | |
| Opti-MEM medium | Gibco, USA | 31985062 | |
| PBS | Gibco, USA | 10010023 | PH 7.4 |
| RNase Inhibitor | Promega, USA | N251B | |
| Streptavidin alkaline phosphatase | Promega, USA | V5591 | |
| TBE | Invitrogen, USA | 15581044 | |
| Tris-HCI Buffer | Invitrogen, USA | 15567027 | 1 M, PH 7.4 |
| Tris-HCI Buffer | Invitrogen, USA | 15568025 | 1 M, PH 8.0 |
| Tween-20 | Sangon Biotech, China | A600560 |
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Citazione di questo articolo
Yu, L., He, W., Xie, J., Guo, R., Ni, J., Zhang, X., Xu, Q., Wang, C., Yue, Q., Li, F., Luo, M., Sun, B., Ye, L., Zheng, K. In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions. J. Vis. Exp. (149), e59830, doi:10.3791/59830 (2019).