无核酸酶的表达和纯化原氧基质 3，4-二氧酶
Summary
原酸3，4-二氧酶（PCD）可以酶分从水系统中分离游离的游离双原子氧，使用其基质原酸（PCA）。该协议描述了这种氧气清除酶的表达、纯化和活性分析。
Abstract
单分子（SM）显微镜用于实时研究荧光体标记生物分子的动态分子相互作用。然而，荧光团容易通过溶解氧（O2）的光漂白失去信号。为了防止光漂白和延长荧光团的寿命，采用氧气清除系统（OSS）来减少O2。市售的OSS可能被破坏或降解核酸的核酸污染，混淆了实验结果的解释。在这里，我们详细介绍了高活性伪单核素的表达和纯化方案，即原氧化酶-3，4-二氧酶（PCD），没有可检测到的核酸酶污染。PCD通过将基质原酸（PCA）转化为3-卡博西-cis-muconic酸，可以有效地去除活性O2种。此方法可用于 O2在数据采集中起到不利作用的任何水性系统。与市售的多氯二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二者相比，该方法能有效地产生高活性、无核酸酶的PCD。
Introduction
单分子（SM）生物物理学是一个迅速成长的领域，它改变了我们对生物现象的观察方式。这一领域具有独特的能力，将物理和化学的基本定律与生物学联系起来。荧光显微镜是一种生物物理方法，可以达到SM灵敏度。荧光用于检测生物分子，将它们与小的有机荧光道或量子点1联系起来。这些分子在光漂白不可逆转之前，被激光激发时可以发射光子。当荧光标签受到化学损伤，破坏其在所需波长2，3处激发的能力时，就会发生光漂白。水缓冲液中活性氧物种（ROS）的存在是光漂白2，4的主要原因。此外，ROS可以破坏生物分子，并导致在SM实验5，6的错误观测。为了防止氧化损伤，氧气清除系统（OSS）可以使用3，7，8。葡萄糖氧化酶/催化酶（GODCAT）系统在去除氧8方面是有效的，但它作为中间体产生潜在的破坏性过氧化物。这些可能损害SM研究中感兴趣的生物分子。
或者，原生化液3，4二氧酶（PCD）将有效地去除O2从水溶液使用其基质原酸（PCA）7，9。PCD是一种金属酶酶，使用非铁来协调PCA，并使用溶解的O2 10催化catechol环开口反应。这一步反应被证明是一个整体更好的OSS，以提高荧光酸稳定性在SM实验7。不幸的是，许多市售的OSS酶，包括多氯二苯并对苯二苯醚，含有污染的核酸酶11。这些污染物可能导致 SM 实验中使用的核酸基板损坏。这项工作将阐明在SM系统中使用重组PCD的基于色谱的纯化方案。PCD 可广泛应用于 ROS 破坏数据采集所需基板的任何实验。
Protocol
1. 诱导大肠杆菌中的多氯二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二苯并对二 在管中结合1 μL pVP91A-pcaHG多氯二苯并对二醇表达质粒（20纳克/μL，图1A）和20 μL大肠杆菌BL21（20 μL市售细胞，>2 x 10 6 cfu/μg质粒）。轻拂管子进行混合。将管子放在冰上 5 分钟。 将变换置于 42°C 30 s。然后冰2分钟。 添…
Representative Results
市售的氧气清除剂PCD经常受到DNA核酸酶的污染。核酸酶活性的污染可能导致荧光研究的虚假结果，特别是分析DNA或DNA相互作用蛋白质的研究。我们发现，重组PCD，六聚二醇标记pcaH和pcaG的异体，可以表达在大肠杆菌（图1）。异质器首先通过镍亲和色谱法进行纯化（图2）。PCD在二分之二浓度的2个步骤中洗脱。色谱分数由SDS-PAGE分…
Discussion
氧气清除系统通常包含在单分子荧光显微镜中，以减少光漂白3，7，8。这些显微镜技术通常用于观察核酸或蛋白质与核酸1、13、14的相互作用。使用核酸酶污染OSS可能导致虚假结果。
商业上可用的开放苷和多氯丁二苯并对二苯并对二苯?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH GM121284和AI126742对KEY的支持。
Materials
| 2-Mercaptoethanol | Sigma-Aldrich | M3148 | βME |
| 30% acrylamide and bis-acrylamide solution, 29:1 | Bio-Rad | 161-0156 | |
| Acetic acid, Glacial Certified ACS | Fisherl Chemical | A38C-212 | |
| Agar, Granulated | BD Biosciences | DF0145-17-0 | |
| AKTA FPLC System | GE Healthcare Life Sciences | AKTA Purifier: Box-900, pH/C-900, UV-900, P-900, and Frac-920 | |
| Amicon Ultra-2 Centrifugal Filter Unit | EMD Millipore | UFC201024 | 10 kDa MWCO |
| Ammonium iron(II) sulfate hexahydrate | Sigma | F-2262 | |
| Ammonium Persulfate (APS) Tablets | Amresco | K833-100TABS | |
| Ampicillin | Amresco | 0339-25G | |
| Bacto Tryptone | BD Biosciences | DF0123173 | |
| BD Bacto Dehydrated Culture Media Additive: Bottle Yeast Extract | VWR | 90004-092 | |
| BIS-TRIS propane,>=99.0% (titration) | Sigma-Aldrich | B6755-500G | |
| Bromophenol Blue | Sigma-Aldrich | B0126-25G | |
| Coomassie Brilliant Blue | Amresco | 0472-50G | |
| Costar 96–Well Flat–Bottom EIA Plate | Bio-Rad | 2240096EDU | |
| DTT | P212121 | SV-DTT | |
| Dulbecco's Phosphate Buffered Saline 500ML | Sigma-Aldrich | D8537-500ML | PBS |
| Ethidium bromide | Thermo Fisher Scientific | BP1302 | |
| Glycerol | Fisher Scientific | G37-20 | |
| Granulated LB Broth Miller | EMD Biosciences | 1.10285.0500 | |
| Hi-Res Standard Agarose | AGTC Bioproducts | AG500D1 | |
| Imidazole | Sigma-Aldrich | I0250-250G | |
| IPTG | Goldbio | I2481C25 | |
| Leupeptin | Roche | 11017128001 | |
| Lysozyme from Chicken Egg White | Sigma-Aldrich | L6876-1G | |
| Magnesium Chloride Hexahydrate | Amresco | 0288-1KG | |
| Microvolume Spectrophotometer, with cuvet capability | Thermo Fisher | ND-2000C | |
| NaCl | P212121 | RP-S23020 | |
| Ni-NTA Superflow (100 ml) | Qiagen | 30430 | |
| Novagen BL21 Competent Cells | EMD Millipore | 69-449-3 | SOC media included |
| Orange G | Fisher Scientific | 0-267 | |
| Pepstatin | Gold Biotechnology | P-020-25 | |
| PMSF | Amresco | 0754-25G | |
| Protocatechuic acid | Fisher Scientific | ICN15642110 | PCA |
| Sodium dodecyl sulfate | P212121 | CI-00270-1KG | |
| SpectraMax M2 Microplate Reader | Molecular Devises | ||
| Sterile Disposable Filter Units with PES Membrane > 250mL | Thermo Fisher Scientific | 09-741-04 | |
| Sterile Disposable Filter Units with PES Membrane > 500mL | Thermo Fisher Scientific | 09-741-02 | |
| Superose 12 10/300 GL | GE Healthcare Life Sciences | 17517301 | |
| TEMED | Amresco | 0761-25ML | |
| Tris Ultra Pure | Gojira Fine Chemicals | UTS1003 | |
| Typhoon 9410 variable mode fluorescent imager | GE Healthcare Life Sciences | ||
| UltraPure EDTA | Invitrogen/Gibco | 15575 | |
| ZnCl2 | Sigma-Aldrich | 208086 |
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Citer Cet Article
Messer, R. K., Lopez Jr., M. A., Senavirathne, G., Yoder, K. E. Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase. J. Vis. Exp. (153), e59599, doi:10.3791/59599 (2019).