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Biochemistry

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Published: October 03, 2018
doi:
10.3791/58307
, , , ,
1Department of Chemistry,Wesleyan University

Summary

在这里, 我们提出了一个协议的厌氧蛋白纯化, 厌氧蛋白浓度, 并随后的动力学表征使用氧电极系统。该方法用酶 DesB, 一种在厌氧环境中纯化和贮存时更稳定、更活跃的酶酶来说明。

Abstract

氧敏感蛋白, 包括那些利用氧作为基质的酶, 可以通过传统的有氧纯化方法, 降低稳定性。本手稿说明了厌氧净化过程中所涉及的技术细节, 包括缓冲剂和试剂的制备、手套盒中柱层析的方法, 以及动力学前蛋白质的脱盐。还介绍了制备和使用氧电极对氧利用酶进行动力学表征的方法。这些方法是使用酶酶 DesB, 一个无食子酶从细菌Sphingobium sp SYK-6。

Introduction

利用铁或其他金属活化氧的酶在纯化过程中往往容易失活, 因为它们从细胞的还原环境中去除。因此, 这些蛋白质必须作为细胞裂解物, 受到外部还原剂, 或纯化厌氧, 以确保他们有最佳的酶活性1,2,3,4。对于那些对氧敏感的酶 (特别是含铁的酶), 执行所有的纯化和表征步骤, 同时保持厌氧条件是充分描述它们的必要条件。这使得研究人员在厌氧室范围内开发了整个实验室, 用于研究从蛋白质表达通过结晶学5,6,7,8.

本文报告了用氧电极系统对酶 DesB 进行厌氧纯化和动力学表征的方法。DesB 是酶细菌Sphingobium菌株 SYK-6 与 LigAB, protocatecuate 酶从同一有机体。这两种酶都属于 II. 型 protocatechuate 酶 (PCAD) 超家族, 至今尚未被广泛研究到9, 可能部分原因是由于这种超家族的酶在纯化使用标准好氧时易失活。蛋白质纯化方法。由于一些 PCAD 酶显示基质混杂, 而另一些是基板特定的2,10, 这一超家族的进一步描述是必要的, 以确定特异性决定因素。正如已经观察到的几个酶 superfamilies11,12,13,14,15, 小分子可以改变活动通过直接竞争抑制或约束分子分离构的口袋, 导致酶活性的增加或减少16。虽然动力学本身不能区分调制器的绑定位置, 但确定活动变化的大小对于理解这些影响是很重要的。因此, 在 4-nitrocatechol (4NC) 的存在下, 本机 DesB 活动的动力学特性及其活性的方法, 通常用来表征和抑制酶酶2,17的化合物,18, 显示。

DesB 可以通过 extradiol 酶 (江户) 反应, 通过氧作为基板10、19的一种催化剂, 来分解木质素衍生的芳香化合物。这种酶反应发生在木质素分解的背景下, 这是植物细胞壁上发现的一种芳香 heteropolymer。木质素可以降解, 产生多种芳香化合物, 可进一步分解成中心代谢产物3,20,21,22,23,24 ,25,26,27,28,29,30,31,32,33.Extradiol dioxygenases (江户) 催化在 dihydroxylated 芳香化合物上的环形开口反应, 其中裂解发生在金属协调二醇附近;相比之下, intradiol dioxygenases 在两个羟基之间进行类似的芳香化合物 (图 1)。EDOs, 像许多其他 metalloenzymes, 有一个价金属中心协调 Fe (II) 由两组氨酸, 一羧酸三联9,34,35。这些 metalloenzymes 变得氧化, 通过氧化或基于机制的失活, 而酶呈现非活动2,36,37,38。

在本手稿所描述的实验过程中, 我们利用 DesB (PCAD) 中的一名来自Sphingobium sp SYK-6 的细菌, 来催化在无食子 C4-C5 键中添加氧气 (图 2A)。这个的 regiochemistry 类似于 LigAB , 这是一个 protocatechuate – 4 , 5 – dioxygenase (图 2B) 。到目前为止, 对这个未食子酶的调查包括没有关于抑制 DesB10、19、39的化合物的报告。利用有氧纯化方法, DesB 表现出多变的活性, 而随着厌氧方法的使用, 我们能够始终如一地获得具有可再生活性的蛋白质。本文介绍的动力学研究显示了 DesB 的厌氧纯化方法、DesB 与未食子酸反应的动力学特性, 以及 4-nitrocatechol (4NC) 对 DesB 的抑制作用。

Protocol

1. 一般材料和方法 按照表 1中的说明准备所有必需的介质。高压釜在120˚C 15 分钟无菌过滤器的 SOC 解决方案, 在添加氯化镁2和葡萄糖后, 通过通过0.2 µm 过滤器。在热处理之前, 调整米勒的溶源性汤 (LB 介质) 溶液的 pH 值。补充 LB-安培介质溶液后, 热处理与无菌溶液的0.2 毫米 l-半胱氨酸, 然后0.1 毫米亚铁铵, 以提高蛋白质的表达和溶解度。 为聚丙烯酰胺凝胶电泳 …

Representative Results

显示的是 DesB-麦芽糖结合蛋白 (MBP) 融合结构纯化的单个组分的 SDS 页凝胶分析 (图 3)。凝胶显示蛋白质是纯净的 (兆瓦 = 91.22 kDa), 除了 DesB 的存在 (兆瓦 = 49.22 kDa) 和 MBP 蛋白质领域 (42 kDa) 互相互相劈开。选择分数 E2 和 E3 浓度 (步骤 4.2)。 DesB 动力学分析的重现性结果取决于正确的装配、校准和实验技术。?…

Discussion

获得活性的、纯化的 DesB 蛋白的关键步骤包括在酶中还原铁 (II.) 活性部位的形成和维持。因此, 正确的诱导、纯化、浓缩和脱盐步骤的性能是成功获得活性酶的必要条件。诱导蛋白在1毫米铁铵存在下的表达确保铁 (II) 正确地纳入 DesB 的活性部位。这种方法是灵感的研究, 如那些与 amidohydrolase metalloenzymes, 这往往需要添加金属的增长媒体, 以允许适当的折叠和充分占用的金属结合点6</sup…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢卫斯理大学的卡米尔. 凯勒博士的技术支持。特别感谢 Eltis 教授和珍娜 k. Capyk 从不列颠哥伦比亚大学, 以及德克萨斯大学奥斯汀分校的基督教惠特曼, 他们就厌氧蛋白质纯化方法和使用 O 2 的建议敏感电极。

Materials

Isopropyl β-D-1-thiogalactopyranodise Gold Bio Technologies I2481C50
Coomassie Brilliant Blue R-250 Bio-Rad 161-0400
Ammonium persulfate Bio-Rad 161-0700
30% Acrylamide Bio-Rad 161-0158
N,N'tetramethyl-ethylenediamine Bio-Rad 161-0801
Amylose Resin High Flow New England Biolabs E8022S
BL21 (DE3) competent Escherichia coli cells New England Biolabs C2527I
L-cysteine Sigma Aldrich C7352
gallic acid Sigma Aldrich G7384
4-nitrocatechol Sigma Aldrich N15553
Ferrous ammonium sulfate Mallinckrodt 5064
Sodium dithionite Alfa Aesar 33381-22
wheaton serum bottles Fisher Scientific 06-406G
25 mm Acrodisc PF Syringe Filter with Supor Membrane Pall Corportation 4187
400 mL Amicon Stirred Cell Concentrator EMD Millipore UFSC40001
76 mm Millipore Ultracel 10 kDa cutoff reconsituted cellulose membrane filter EMD Millipore PLGC07610
DL-dithiothreitol Gold Bio Technologies DTT50
Sephadex G-25 coarse desalting gal column GE Healthcare 17-0033-01
2 mL Crimp-Top Vials Fisher Scientific 03-391-38
Oxygraph Plus Electrode Control Unit Hansatech Instruments OXYG1 Plus
Oxygen Eletrode Chamber Hansatech Instruments DW1
Electrode Disc Hansatech Instruments S1
PTFE (0.0125 mmX25mm) 30m reel Hansatech Instruments S4
Electrode cleaning Kit Hansatech Instruments S16
Spacer paper Zig Zag available at any gas station
He-series Dri-Lab glove box Vacuum/Atmospheres Company
HE-493 Dri-Train Vacuum/Atmospheres Company
Double-Ended Micro-Tapered Stainless Steel Spatula Fisher Scientific 21-401-10
DWK Life Sciences Kimble Kontes Flex Column Economy Column Fisher Scientific k420400-1530
10 μL, Model 701 N SYR, Cemented NDL 26s ga, 2 in, point stlye 2 syringe Hamilton 80300
DWK Life Sciences Kimble Kontes Flex Column Economy Column Fisher Scientific K420401-1505
Emulsiflex-C5 high-pressure homogenizer Avestin
B-PER Complete Bacterial Protein Extraction Reagent Thermo Fisher Scientific 89821
Lysozyme from chicken egg white Sigma Aldrich 12650-88-3
Sodium dodecyl sulfate Thermo Fisher Scientific 151-21-3
ampicillin Sigma Aldrich 7177-48-2
Tryptone Fisher Scientific BP-1421-500
Yeast extract Fisher Scientific BP1422-2
Sodium Chloride Fisher Scientific S271-10
Potassium Chloride Fisher Scientific P217-3
Magnesium Chloride Fisher Scientific M33-500
Dextrose Fisher Scientific D16-3
Sodium Hydroxide Fisher Scientific S318-1
Tris hydrochloride Fisher Scientific BP153-500
Maltose Fisher Scientific BP684-500
Glycine Fisher Scientific G46-500
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AnaerobicProtein PurificationKinetic AnalysisOxygen ElectrodeDesB DioxygenaseEnzyme ActivityInhibitionInorganic BiochemistryOxygen-sensitive MetalsProtein ExpressionCell PelletAmylose ColumnLysozymeHigh-pressure HomogenizationNitrogen GasGlove BoxAmylose Column BufferElution BufferFerrous Ammonium SulfateDithiothreitol
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Uchendu, S. N., Rafalowski, A., Cohn, E. F., Davoren, L. W., Taylor, E. A. Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition. J. Vis. Exp. (140), e58307, doi:10.3791/58307 (2018).
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