Microscale Two-Stage Liquid-Liquid Extraction for Purification of Engineered Proteins

We have developed a process for micro-preparative purification of nanogram quantities of engineered proteins. An aqueous two-phase system consisting of polyethylene glycol and potassium phosphate is used to perform a laminar flow two phase extraction on a chip. During the first stage of extraction, an aromatic-rich “partition tag” at the N-terminus of a target protein drives partitioning into the PEG-rich phase of the two-phase system. In between stages, a protease (thrombin) is used for site-specific cleavage of the partition tag, which causes a reversal in the partitioning behavior of the target protein, without affecting other proteins in the mixture. A second stage of laminar flow extraction is performed on chip to back-extract the cleaved protein into the salt-rich phase, away from proteins that had co-partitioned to the PEG phase during the first stage of extraction. The process is mild enough to maintain the structure and function of a fluorescent protein. The two-stage process results in improved purity versus a single-stage process previously reported. Two-phase extraction is an alternative to affinity chromatography for very small amounts of protein. A major advantage of the process is the simplicity of the device, which features no moving parts and no chromatography resins or supports.