蓝藻中完整藻胆体的分离与表征
Summary
目前的方案详细介绍了通过不连续蔗糖密度梯度离心从蓝藻中分离藻胆体的过程。完整的藻糖体的分数通过77K荧光发射光谱和SDS-PAGE分析得到证实。所得的藻胆体组分适用于TEM的负染色和质谱分析。
Abstract
在蓝藻中，藻胆体是一种重要的天线蛋白复合物，它收集光并将能量转移到光系统I和II进行光化学。研究藻胆体的结构和组成对科学家来说非常有趣，因为它揭示了蓝藻中光合作用的进化和分化。该协议提供了一种详细且优化的方法，通过磁珠打浆机以低成本有效地破坏蓝藻细胞。然后，完整的藻胆体可以通过蔗糖梯度超速离心从细胞提取物中分离出来。该方法已被证明适用于具有不同细胞类型的模型和非模型蓝藻。还提供了分步程序，通过77K荧光光谱和SDS-PAGE用硫酸锌和考马斯蓝染色来确认藻胆蛋白的完整性和性质。分离出的藻胆体也可以进行进一步的结构和成分分析。总体而言，该协议提供了一个有用的入门指南，使不熟悉蓝藻的研究人员能够快速分离和表征完整的藻类化合物。
Introduction
藻胆体（PBS）是一种巨大的水溶性色素 – 蛋白质复合物，附着在蓝细菌的类囊体膜中光系统的细胞质侧1。PBS主要由有色藻胆蛋白和无色连接蛋白组成1，2。藻胆蛋白可分为四大类：藻红素、藻红蓝蛋白、藻蓝蛋白和异体花青素3。四大类吸收490-650nm范围内不同波长的光能，叶绿素吸收效率低下3。PBS可以用作光收集天线，用于收集光能并将其输送到光系统II和I4。
PBS的结构和组成因物种而异。总的来说，在不同的蓝藻物种中已经鉴定出三种形状的藻胆体（半盘状、束状和杆状）5。即使在同一物种中，PBS的组成也会随环境而变化，例如光质和营养消耗6，7，8，9，10，11。因此，从蓝藻中分离PBS的实验程序有助于研究PBS12。几十年来，许多不同的方案已经分离出PBS并分析了其结构，组成和功能6，7，8，12，13，14，15，16，17。PBS分离方法种类繁多，确实为使用不同的试剂和仪器分离不同物种中的复合物提供了灵活性。然而，这也使得不熟悉蓝藻和PBS的科学家更难选择合适的方案。因此，在这项工作中为那些有兴趣从蓝藻中开始PBS分离的人开发了一个通用且直接的方案。
此处总结了从以前的出版物中分离 PBS 的方法。由于PBS是一种水溶性蛋白质复合物并且容易解离，因此在提取过程中需要高离子强度的磷酸盐缓冲液来稳定PBS18。过去曾发表过几篇描述从蓝藻中分离PBS的方法的研究文章。大多数方法需要高浓度的磷酸盐缓冲液8，14，15，18，19。然而，机械破坏细胞的程序各不相同，例如玻璃珠辅助提取，超声处理20和法式压榨6，8，14。不同的藻胆蛋白可以通过用硫酸铵20 沉淀并通过HPLC21 或色谱柱22纯化来获得。另一方面，完整的PBS可以通过蔗糖密度梯度超速离心轻松分离6，8，15。
在该协议中，使用一种模型蓝藻和一种非模型蓝藻作为PBS分离的材料。它们是模型单细胞葡萄糖耐受 性Synechocystis sp. PCC 6803（以下简称Syn6803）和非模型丝状 Leptolyngbya sp. JSC-1（以下简称JSC-1），分别7，23，24。该协议首先在高离子强度磷酸盐缓冲液中破坏单细胞和丝状蓝藻。裂解后，通过离心收集上清液，然后用非离子洗涤剂（Triton X-100）处理，以溶解来自类囊体膜的水溶性蛋白质。将总水溶性蛋白施加到不连续的蔗糖密度梯度上以分馏PBS。该方案中不连续的蔗糖梯度由四种蔗糖溶液组成，并将完整的PBS分配在蔗糖层的最低部分25中。PBS的完整性可以通过SDS-PAGE，锌染色和77K荧光光谱分析6，7，8，26。这种方法适用于旨在从蓝藻中分离完整PBS并研究其光谱，结构和组成特性的科学家。
此协议有几个优点。（1）该方法是标准化的，可用于从单细胞和丝状蓝藻中分离完整的PBS。大多数文章描述了应用于任何一种类型的蓝藻4，7，8，12，13，14，16，18的方法。（2）该方法在室温下进行，因为PBS在低温下解离19，27。（3）该方法描述了使用珠状敲击剂来破坏细胞;因此，它比高压法国压力机更便宜，更安全，并且其他方法中超声波仪可能造成的听力损伤8，13，14，20。（4）该方法通过蔗糖梯度超速离心分离完整的PBS。通过这种方式，可以根据蔗糖浓度分离具有不同大小和部分解离PBS的完整PBS。
Protocol
胞藻属PCC 6803，模型葡萄糖耐受菌株，从台湾中央研究院的Chu，Hsiu-An博士获得。Leptolyngbya sp. JSC-1，非模型丝状，是从美国宾夕法尼亚州立大学的Donald A. Bryant博士那里获得的。 1. 细胞培养和收获 使用金属接种回路将Syn6803或JSC-1细胞接种到含有50 mL B-HEPES培养基的100 mL锥形烧瓶中28。在30°C下，在50μmol光子m-2 s-1（白…
Representative Results
将Syn6803和JSC-1细胞在锥形烧瓶中培养，在30°C的B-HEPES培养基中，在LED白光（50μmol光子m-2s-1）下在充满1%（v / v）CO2的生长室中持续搅拌。在指数生长阶段（OD750 = ~0.5），将细胞转培养到最终光密度为OD 750 = ~0.2的新鲜培养基中。在达到晚期指数生长阶段（OD750 = 0.6-0.8）后，收集培养物并离心（室温下10，000×g20分钟）。弃去上清液，并将细胞沉…
Discussion
该协议描述了一种简单而标准的方法，用于分离两种类型的蓝藻中的完整PBS，单细胞模型Syn6803和丝状非模型JSC-1。该方案的关键步骤是在蔗糖的不连续密度梯度上进行细胞均质化和超速离心。通常，丝状细胞的破坏比单细胞细胞更复杂。增加起始物质的量（细胞沉淀的湿重）和重复的磁珠打有助于提高丝状蓝藻细胞PBS的产量。对于丝状蓝藻JSC-1，使用的细胞是单细胞Syn6803的三倍。此外，要完全破?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢国立台湾大学生命科学学院Technology Commons对超速离心机的方便使用。蓝藻菌株 Synechocystis sp. PCC 6803和 Leptolyngbya sp. JSC-1分别由台湾中央研究院的Chu，Hsiu-An博士和美国宾夕法尼亚州立大学的Donald A. Bryant博士赠送。这项工作由科学技术部（台湾）（109-2636-B-002-013-和110-2628-B-002-065-）和教育部（台湾）玉山青年学者计划（109V1102和110V1102）资助。
Materials
| 0.1 mm glass beads | BioSpec | 11079101 | for PBS extraction |
| 13 mL centrifugation tube | Hitachi | 13PA | ultracentrifugation |
| 40 mL centrifugation tube | Hitachi | 40PA | ultracentrifugation |
| Acetic acid | Merck | 8.1875.2500 | for Coomassie Blue staining |
| B-HEPES medium | A modified cyanobacterial medium from BG-11 medium | ||
| Brilliant Blue R-250 | Sigma | B-0149 | for Coomassie Blue staining |
| Bromophenol blue | Wako pure chemical industries | 2-291 | protein loading buffer |
| Electronic balance | Radwag | WLC 2/A2/C/2 | for the wet weight measurement of cell pellets |
| Fluorescence spectrophotometer | Hitachi | F-7000 | Spectrophotometer |
| Glycerol | BioShop | Gly001.500 | protein loading buffer |
| High-Speed refrigerated centrifuge | Hitachi | CR22N | for buffer exchange |
| Leptolyngbya sp. JSC-1 | from Dr. Donald A. Bryant at Pennsylvania State University, USA. | ||
| Low temperature measurement accessory | Hitachi | 5J0-0112 | The accessory includes a transparent Dewar container for 77K fluorescence spectra |
| Methanol | Merck | 1.07018,2511 | for Coomassie Blue staining |
| Microcentrifuge | Thermo Fisher | Pico 21 | for PBS extraction |
| Mini-Beadbeater-16 | BioSpec | Model 607 | for PBS extraction |
| Potassium phosphate dibasic | PanReac AppliChem | 121512.121 | for PBS extraction |
| Potassium phosphate monobasic | PanReac AppliChem | 141509.121 | for PBS extraction |
| Screw cap vial | BioSpec | 10832 | for PBS extraction |
| SmartView Pro Imager | Major Science | UVCI-2300 | for Znic staining signal detection |
| Sodium dodecyl sulfate | Zymeset | BSD101 | protein loading buffer |
| Sucrose | Zymeset | BSU101 | for PBS isolation |
| Synechocystis sp. PCC 6803 | glucose-tolerant strain from Dr. Chu, Hsiu-An at Academia Sinica, Taiwan | ||
| Tris | BioShop | TRS 011.1 | protein loading buffer |
| Triton X-100 | BioShop | TRX 506.500 | for PBS extraction |
| Ultra 10 K membrane centrifugal filter | Millipore | UFC901024 | for buffer exchange |
| Ultra 3 K membrane centrifugal filter | Millipore | UFC500324 | for buffer exchange |
| Ultracentrifuge | Hitachi | CP80WX | ultracentrifugation |
| UV/Vis spectrophotometer | Agilent | Cary 60 | Spectrophotometer |
| Zinc sulfate | PanReac AppliChem | 131787.121 | for Znic staining |
| β-Mercaptoethanol | BioBasic | MB0338 | protein loading buffer |
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Jiang, H., Ho, M. Isolation and Characterization of Intact Phycobilisome in Cyanobacteria. J. Vis. Exp. (177), e63272, doi:10.3791/63272 (2021).