Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags

Abstract

High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes approximately 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.

On-chip immunoprecipitation for protein purification

“…In nearly all reports to date, model mixtures have been used to demonstrate protein enrichment and very little has been reported on the separation of complex biological samples. An exception is the work by Meagher et al, 20 where recombinant proteins from an Escherichia coli lysate were enriched within a PEG-rich phase through use of a hydrophobic partition tag.…”

“…[17][18][19][20] These include bead-based affinity methods, where beads with an appropriate surface functionality are packed into a microfluidic channel, and the partitioning of proteins using two-phase flow extraction methods. In nearly all reports to date, model mixtures have been used to demonstrate protein enrichment and very little has been reported on the separation of complex biological samples.…”

Tunable spatial heterogeneity in structure and composition within aqueous microfluidic droplets

“…The partitioning of the polymer mixtures into different thermodynamic phases within such encapsulated structures is analogous to micro-compartmentalization in living cells. 6 Microfluidics-based applications of ATPS, such as fabrication of hydrogel beads, 7 nanolitre liquid patterning, 8 and separation of dyes, 1 cells, [9][10][11][12][13] and proteins 14,15 have been demonstrated in recent years. Several applications involve droplet-based microfluidics, and two typical droplet morphologies are used: "single emulsions" where one aqueous polymer droplet is dispersed in another aqueous polymer solution that serves as the continuous phase 1,7 or "double emulsions" where one aqueous polymer droplet is completely engulfed within another aqueous polymer a) Author to whom correspondence should be addressed.…”

All-aqueous multiphase microfluidics

“…Manipulation of all-aqueous jets in microfluidic devices has been shown to be a critical tool in the separation of DNA, 16,17 proteins, 18,19 and cells. 20 In comparison to water/oil systems, common ATPSs have much lower interfacial tension values, ranging from less than 10 À2 mN/m to 1 mN/m (Table II).…”