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Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system
Journal
Journal of Chromatography A
Volume
1618
Pages / Article-Number
460846
Keywords
ALIAS autosampler, automated protein purification, in-line sample dilution, ion-exchange chromatography, Green Fluorescent Protein, G protein-coupled receptors
Protein purification processes in basic research using ÄKTA TM liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS TM autosampler and a modular sample in-line dilution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion exchange to size exclusion and nickel-ion metal affinity to size exclusion. The chromatographic setup enabled purification of a large array of cytosolic and membrane proteins from small-scale expression cultures produced in insect cells necessary to develop and optimize isotope-labeling strategies for nuclear magnetic resonance spectroscopy applications, resulting in a reduction in experiment time of about 20% per run for both cytosolic and membrane protein purification schemes. However, when queuing multiple samples the throughput increased by 66% and 75%, respectively. In addition, a novel system configuration is presented, where two column valves can be operated independently. This allows for the design of purification loops to increase purity of the target protein.
