向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质
Summary
这里描述了分离蓝藻释放的碳水化合物聚合物和分离其外蛋白酶的协议。这两种程序都体现了获得高纯度聚合物或蛋白质的关键步骤,可用于进一步分析或应用。它们也可以根据特定的用户需求轻松调整。
Abstract
蓝藻可以主动将多种生物分子分泌到细胞外环境中,如异质多糖和蛋白质。这些生物分子的鉴定和表征可以增进有关其分泌途径的知识,并有助于操纵它们。此外,其中一些生物分子在生物技术应用方面也很有趣。这里描述了两个协议,用于轻松和快速分离蓝藻释放的碳水化合物聚合物和蛋白质。释放的碳水化合物聚合物的分离方法基于使用有机溶剂在水溶液中多糖的传统沉淀技术。这种方法保留了聚合物的特性,同时避免了来自细胞碎片和培养基培养物的污染物的存在。在工艺结束时,冻干聚合物可供使用或定性,或可以进行进一步轮纯化,具体取决于最终预期用途。关于蓝藻外蛋白酶的分离,该技术基于通过离心和过滤去除主要污染物后无细胞介质的浓度。这种策略允许通过膜转运器或外膜囊泡可靠地分离通过细胞外环境的蛋白质。这些蛋白质随后可以使用标准质谱技术进行识别。这里提出的协议不仅适用于广泛的蓝藻,而且还适用于其他细菌菌株。此外,这些程序可以很容易地根据产品的最终用途、所需的纯度和细菌菌株进行定制。
Introduction
蓝藻被广泛认为是天然产品的丰富来源,具有广阔的生物技术/生物医学应用。因此,了解蓝藻分泌机制和优化提取/回收方法对于将蓝藻作为高效的微生物细胞工厂实施至关重要。
许多蓝藻菌株能够产生细胞外聚合物物质(EPS),主要由异聚糖形成,这些物质仍然与细胞表面相关或释放到介质1中。与其他细菌相比,这些释放的碳水化合物聚合物具有明显的特性,这使得它们适用于广泛的应用(例如,抗病毒药物2、免疫刺激3、抗氧化剂4、金属夹合5,乳化6,和药物输送剂7,8。分离这些聚合物的方法不仅有助于提高产量,而且有助于提高纯度,提高聚合物获得的具体物理特性。绝大多数这些方法分离聚合物依赖于沉淀策略从培养基,很容易完成由于聚合物的强烈电子性质9,10。此外,通过蒸发和/或冻干,可以迅速去除沉淀步骤中使用的溶剂。根据所预见的应用,在聚合物沉淀之后或之前可以耦合不同的步骤,以便定制最终产品,包括三氯乙酸 (TCA) 处理、过滤或尺寸排除色谱 (SEC)列净化10.
蓝藻也能够通过依赖膜输送器(经典)11或囊泡(非经典)12的通道分泌多种蛋白质。因此,分析蓝藻外蛋白体是了解/操纵蓝藻蛋白分泌机制和了解这些蛋白质的具体细胞外功能的重要工具。对外蛋白酶的可靠分离和分析需要细胞外环境的浓度,因为分泌蛋白质的丰度相对较低。此外,其他物理或化学步骤(例如离心、过滤或蛋白质沉淀)可以优化获得的外乳激素的质量,丰富蛋白质含量13,并避免污染物的存在(例如,色素、碳水化合物等)14,15或细胞内蛋白质在样本中的优势。然而,其中一些步骤也可能限制可以检测到的蛋白质集,从而导致有偏差的分析。
本作品描述了从蓝藻培养培养物中分离释放的碳水化合物聚合物和外丙酮的有效方案。这些协议可以很容易地适应研究的具体目标和用户需求,同时保持此处介绍的基本步骤。
Protocol
Representative Results
Discussion
为了更好地了解细菌分泌机制和研究已释放的产品,证明在细胞外细菌环境中(如释放)中存在的生物分子的有效分离和分析具有极其重要的意义。碳水化合物聚合物和蛋白质)。
青基体细胞外碳水化合物聚合物极其复杂,主要是由于构成其成分1的不同单糖的数量和比例。用于分离这些聚合物物质的传统方法依赖于一个简单的概念,即这些富含糖的物质可溶于水溶?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作由欧洲发展基金会(FEDER)通过竞争-竞争力和国际化歌剧方案(POCI),葡萄牙2020年,葡萄牙基金通过FCT- 基金会资助a 在POCI-01-01-FEDER-028779项目框架内,在项目POCI-01-0145-FEDER-028779和赠款SFRH/BD/99715/2014(CF)中,技术技术/Ministério da Cióncia,Tecnologia e Ensino 高级版。
Materials
| Dialysis membranes | Medicell Membranes Ltd | DTV.12000.07 | Visking Tubing Size 7, Dia 23.8 mm, Width 39-41 mm 30m Roll |
| Ethanol 96% | AGA – Álcool e Géneros Alimentares, S.A. | 4.000.02.02.00 | Fermentation ethyl alcohol 96% AGA |
| PES Filter 0.2 μm | Fisher Scientific, Lda | 15206869 | Syringe filter polystyrene 33MM 0.2µM STR |
| Amicon Ultra-15, Ultracel-3K | Merck Millipore Ltd. | UFC900324 | Centrifugal filters with a nominal molecular weight cut-off of 3 kDa |
| Thermo Scientific Pierce BCA Protein Assay | Fisher Scientific, Lda | 10741395 | Green-to-blue, precise, detergent-compatible assay reagent to measure total protein concentration |
| Brillant Blue G Colloidal Concentrate | Sigma Aldrich Química SL | B2025-1EA | Coomassie blue |
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