Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-580 Santo André, SP, Brazil.
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-580 Santo André, SP, Brazil.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Nov 1;1130-1131:121806. doi: 10.1016/j.jchromb.2019.121806. Epub 2019 Oct 4.
Lectins are carbohydrate binding proteins with many physiological and biotechnological applications. Isolation of proteins is normally time-consuming and encompasses multiple and, sometimes, complicated steps that hinder reproducibility and yield. Affinity chromatography is an efficient way to simplify and improve protein purification, however often requiring an expensive and fragile stationary phase. In this regard, automated flow-based systems minimize the time for extraction of species from solid samples without hindering the features of batch procedures. In this work, a new inexpensive affinity-based stationary phase was developed for in-line separation of jacalin, a galactose-binding lectin from jackfruit seeds. In the flow manifold, in-line extraction of proteins was also carried out with continuous monitoring using the spectrophotometric Biuret assay. For protein determination, linear response was observed from 3.0 to 15 g L. The results of the analysis of protein extracts from jackfruit seeds obtained with the herein described procedure and batch procedure agreed with 95% confidence level. Quantitative extraction of proteins from jackfruit seed powder required recirculation of extraction buffer for 15 min through a lab-made polymethylmethacrylate (PMMA) column containing 200 mg of the crude seed powder. In the chromatographic step, jacalin was isolated after 300 s. Therefore, three essential steps for jacalin isolation were performed in one manifold in a fast way, minimizing sample consumption and solution handling. Additionally, the versatile and multi-task developed flow manifold can be useful for routine analysis and preparative procedures, being adaptable for the extraction and separation of other species from solid matrixes with continuous monitoring of the processes.
凝集素是具有许多生理和生物技术应用的碳水化合物结合蛋白。蛋白质的分离通常很耗时，并且包含多个，有时甚至是复杂的步骤，这些步骤会影响重现性和产量。亲和层析是简化和改进蛋白质纯化的有效方法，但是通常需要昂贵且易碎的固定相。在这方面，自动化的基于流的系统可以最大程度地减少从固体样品中提取物种的时间，而不会阻碍批量程序的特征。在这项工作中，开发了一种新的廉价亲和固定相，用于从 jackfruit 种子中的半乳糖结合凝集素 jacalin 进行在线分离。在流路中，还通过使用分光光度比色法 Biuret 测定法进行连续监测来进行在线蛋白质提取。对于蛋白质测定，从 3.0 到 15 g/L 观察到线性响应。用本文所述程序和批处理程序从 jackfruit 种子中提取蛋白质提取物的分析结果在 95%置信水平下一致。从 jackfruit 种子粉末中定量提取蛋白质需要将提取缓冲液通过实验室自制的聚甲基丙烯酸甲酯（PMMA）柱循环 15 分钟，该柱含有 200 mg 的粗种子粉末。在色谱步骤中，jacalin 在 300 s 后被分离。因此，在一个流路中以快速的方式完成了 jacalin 分离的三个基本步骤，从而最大程度地减少了样品消耗和溶液处理。此外，开发的多功能多任务流路可以用于常规分析和制备程序，可用于从固体基质中提取和分离其他物质，并连续监测过程。
