分光光度法测定蓝藻Synechocystis中的蛋白含量
Summary
在这里, 我们提出了一个协议, 以定量地确定蛋白的内容在蓝藻Synechocystis使用分光光度法。提取过程也成功地应用于其他蓝藻和藻类菌株;然而, 由于颜料吸收谱的变化, 有必要分别测试每种菌株的分光光度方程。
Abstract
这是一个简单的协议, 以定量测定蛋白的内容在模型蓝藻Synechocystis。藻胆蛋白是 phycobilisomes 中最重要的成分, 是蓝藻中主要的光捕获触角和几种藻类群。Synechocystis的 phycobilisomes 包含两个藻胆蛋白: 藻和复方阿司匹林。该协议描述了一种简单、高效、可靠的方法, 用于定量测定该模型蓝藻中的藻和复方阿司匹林。比较了几种蛋白提取方法和分光光度法。本协议中所述的萃取过程也成功地应用于其他蓝藻菌株, 如Cyanothece 、 Synechococcuselongatus、螺旋藻、节旋藻sp、和念珠菌, 以及红藻类紫球藻藻。然而, 不同分类群的特定藻胆蛋白的消光系数可能不同, 因此建议分别验证每一个单株的分光光度法。该议定书需要很少的时间, 可以在任何标准生命科学实验室进行, 因为它只需要标准设备。
Introduction
fPhycobiliproteins 是水溶性色素蛋白复合物, 代表原核蓝藻 (蓝藻) 和几种真核生物群中的光收获触角的主要成分 (Glaucophyta、红藻门和Cryptophyta)1。它们主要发生在称为 phycobilisomes 的超分子复合体上, 通常附着在基质侧的光合膜表面, 除Cryptophyta外, 藻胆蛋白在囊体腔2。迄今已确定了四种类型的藻胆蛋白: 核心复方阿司匹林和外围藻、藻红蛋白和 phycoerythrocyanin1。phycobilisomes 作为主要的光收获配合物, 是藻类和蓝藻群体培养生产力的重要因素之一。结果表明, phycobilisomes 截断可提高强光下3的生物量积累。另一方面, 在适度或低辐照度下, 天线截断导致生长速率和生物量积累减少3,4。藻胆蛋白在化妆品行业中作为食品着色剂、药品和食品添加剂, 并作为荧光探针应用于流式细胞术、荧光免疫和荧光显微镜5。
本协议着重于蓝藻Synechocystis模型中藻胆蛋白的定量测定。蓝藻是最早的氧光合自养;他们已经形成了地球的生物圈超过24亿年6。它们在氮、碳、氧和其他元素的全球生物地球化学循环中起着至关重要的作用。在蓝藻中, 单细胞菌株Synechocystis获得了独特的地位, 因为它是第一个蓝藻与整个基因组序列7,8, 它是自然转化的外源 DNA9, 并它执行稳定和相对地快速的成长10,11。在Synechocystis, 核心天线组件, 复方阿司匹林, 是与整体膜蛋白, 并附上藻位于囊体膜外围。
在该协议中比较了蛋白提取和定量的几种方法。最后提取程序成功地应用于Synechocystis, 以及其他蓝藻菌株, 包括Cyanothece sp, Synechococcuselongatus,螺旋藻sp,节旋藻sp和念珠菌, 也成功地应用于红藻类紫球藻藻。因此, 本协议中开发的方法可以被认为是蛋白提取的通用方法。尽管一些经过测试的提取方法导致了总蛋白产量的提高, 这里描述的萃取过程提供了最高的蛋白产量连同叶绿素的最低含量的残留在蛋白提取物。降低叶绿素a的含量是正确藻和复方阿司匹林分光光度定量的关键。
不同藻类和蓝藻物种的蛋白吸收光谱在12、13、14、15、16、17 、甚至在18种单一蓝藻属的几种菌株中。因此, 用于测定Synechocystis中藻和复方阿司匹林的特定波长和吸收系数一般不适用于其他菌株。此外, Synechocystis不包含藻红蛋白和 phycoerythrocyanin, 可以在其他藻类和蓝藻中发现。为了确定非Synechocystis菌株中的藻胆蛋白, 建议分别对各菌株的分光光度方程进行评价。
虽然该议定书包含两个较长的步骤 (夜间冷冻干燥的细胞颗粒和1小时蛋白质提取), 总的劳动时间为藻胆蛋白量化不超过2小时。
Protocol
1. 蓝藻栽培 培养Synechocystis细胞在锥形烧瓶或光生物反应器10,19在缓冲 BG11 中等20保持 pH < 10 (例如, 使用17毫米 HEPES10)。注: 标准栽培条件要求受控温度 (通常为30摄氏度, 最佳温度为35°c)21, 光照 (通常是强度高达800µmol [光子]/[m2·s])21的白色光, 和 …
Representative Results
对于最初的方法测试, Synechocystis被培养了作为批培养在锥形烧瓶在 BG11 耕种媒介20 (补充与17毫米 HEPES) 在25°c, 在一个温暖的白色光之下强度的50µmol (光子)/(m2·s) 和与 1% CO2在培养的大气。在养殖过程中, 将这些培养物取样到安全锁管和离心 (实验室温度为5分钟 1.5万克), 将清液丢弃, 并将样品储存在-80 摄氏度, 冷冻干燥作为制?…
Discussion
该协议描述了一种简单、快速、可重现的方法, 用于量化蓝藻Synechocystis中的蛋白内容。比较了细胞均匀化、蛋白质提取、藻和复方阿司匹林量化的几种方法, 最后的协议代表了每一个过程的最优步骤的组合。作为代表性数据, 藻胆蛋白在Synechocystis细胞中的含量随着光照强度的增加而定量化。尽管分析需要类似的时间和实验室设备作为一些以前发布的方法12,<su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该议定书是从上一个出版物11通过的。t.z.、d. Č和 J. 由捷克共和国教育、青年和体育部在国家可持续发展方案 (NPU 一) 的支持下, 授予编号 LO1415。J. Č也得到了 GA CR 的支持, 18-24397S 号。捷克的系统生物学 C4SYS 研究基础设施 (项目没有 LM2015055) 支持获得仪器和其他设施的机会。m.a.s. 得到了俄罗斯科学基金会的资助 [14-14-00904]。
Materials
| Synechocystis sp. PCC 6803 | Institut Pasteur, Paris, France | 6803 | Cyanobacterium strain |
| Roti-CELL PBS | Carl Roth GmbH + Co. KG, Karlsruhe, Germany | 9143.1 | Phosphate-Buffered Saline (PBS) solution, pH 7.4 |
| Eppendorf safe-lock tubes | Eppendorf, Hamburk, Germany | 30120086 | Safe-lock tubes 1.5 ml |
| VWR 80-Place Storage System | VWR International, Radnor, Pennsylvania, USA | 30128-282 | Holder for safe-lock tubes |
| RAININ 100 µl -1000 µl | Mettler-Toledo, Columbus, Ohio, USA | 17014382 | Pipette |
| GP-LTS-A-1000µL-/F-768/8 | Mettler-Toledo, Columbus, Ohio, USA | 30389272 | Pipette tips |
| Rotina 420R | Hettich, Kirchlengern, Germany | 4701 | Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes |
| LCexv 4010 | Liebherr, Bulle, Switzerland | 9005382197172 | Refrigerator and freezer -20 °C |
| Revco ExF -86°C Upright Ultra-Low Temperature Freezer | Thermo Fisher Scientific, Waltham, Massachusetts, USA | EXF24086V | Freezer -80 °C |
| CoolSafe | LaboGene, Lillerød, Denmark | 7.001.000.615 | Freeze dryer |
| UV-2600 | Shimadzu, Kyoto, Japan | UV-2600 | Spectrophotometer |
| Hellma absorption cuvettes, semi Micro | Sigma-Aldrich, St. Louis, Missouri, USA | Z600288 | VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm |
| Silamat S6 | Ivoclar Vivadent, Schaan, Liechtenstein | 602286WU | Homogenizer |
| Solid-glass beads | Sigma-Aldrich, St. Louis, Missouri, USA | Z273627 | Glass bead of the diameter 2 mm |
| CPA225D-0CE | Sartorius AG, Göttingen, Germany | SECURA225D-1OBR | Analytical balances |
| C-Phycocyanin from Spirulina sp. | Sigma-Aldrich, St. Louis, Missouri, USA | P2172 | Phycocyanin standard |
| Allophycocyanin | Sigma-Aldrich, St. Louis, Missouri, USA | A7472 | Allophycocyanin standard |
| Bicinchoninic Acid Kit | Sigma-Aldrich, St. Louis, Missouri, USA | BCA1, B9643 | Complete kit for total proteins determination |
| AlgaeTron | Photon System Instruments Ltd., Drásov, Czech Republic | AG 130-ECO | Cultivation chamber for E. flasks, with controllable light and atmosphere |
| Photobioreactor | Photon System Instruments Ltd., Drásov, Czech Republic | FMT-150 | Cultivation equipment for cyanobacteria and algae with completely controllable environment |
| Cellometer | Nexcelom Bioscience, Lawrence, Massachusetts, USA | Auto M10 | Cell counter |
| Corning 15 mL centrifuge tubes | Sigma-Aldrich, St. Louis, Missouri, USA | CLS430791 | 15 ml Centrifuge tube for dry weigth sampling |
| Herasafe KS | Thermo Fisher Scientific, Waltham, Massachusetts, USA | 51024579 | Laminar flow hood |
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Cite This Article
Zavřel, T., Chmelík, D., Sinetova, M. A., Červený, J. Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis. J. Vis. Exp. (139), e58076, doi:10.3791/58076 (2018).