Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system

Area of Science:

• Biochemistry
• Structural Biology
• Chromatography

Background:

• Standard protein purification methods using ÄKTA™ systems are often manual, single-sample, and limited to one-column protocols.
• Complex purification strategies for structural biology targets can be laborious and time-consuming.
• There is a need for automated and streamlined workflows to accelerate protein production.

Purpose of the Study:

• To automate common protein purification strategies, specifically ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion.
• To reduce the time and labor associated with purifying diverse proteins for applications like nuclear magnetic resonance (NMR) spectroscopy.
• To enhance the throughput and purity of target proteins from small-scale expression cultures.

Main Methods:

• Incorporation of an ALIAS™ autosampler and a modular in-line dilution system into the chromatography workflow.
• Automation of two common purification schemes: ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion.
• Implementation of a novel system configuration with two independently operated column valves for purification loops.

Main Results:

• Achieved a 20% reduction in experiment time per run for both cytosolic and membrane protein purification.
• Increased throughput by 66% and 75% when queuing multiple samples.
• Demonstrated successful purification of various cytosolic and membrane proteins from insect cell expression for NMR applications.

Conclusions:

• The automated workflow significantly streamlines downstream protein production, making it more efficient.
• The system enables the purification of a wide range of proteins required for developing and optimizing isotope-labeling strategies for NMR.
• The novel column valve configuration allows for enhanced protein purity through purification loops.