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. 2019 Nov 1;1130-1131:121806.
doi: 10.1016/j.jchromb.2019.121806. Epub 2019 Oct 4.

An Automated Flow System for In-Line Extraction of Proteins and Separation of Jacalin Using a Low-Cost Stationary Phase

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An Automated Flow System for In-Line Extraction of Proteins and Separation of Jacalin Using a Low-Cost Stationary Phase

Rodrigo S N Mancini et al. J Chromatogr B Analyt Technol Biomed Life Sci. .

Abstract

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-1. 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.

Keywords: Affinity chromatography; Automation; Purification; Solid-liquid extraction.

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