title: 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.
authors: Sandia National Laboratories, Biosystems Research Department, P.O. Box 969, Livermore, CA 95391, USA.
authors: Robert J
authors: RJ
authors: Meagher
authors: Yooli K
authors: YK
authors: Light
authors: Anup K
authors: AK
authors: Singh
mesh_terms: D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D005818:Genetic Engineering / Q000379:methods*; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D049408:Luminescent Agents / Q000737:chemistry / Q000302:isolation & purification; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012996:Solutions / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D049408:Luminescent Agents; D011506:Proteins; D012996:Solutions; D014867:Water; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D005982:Glutathione Transferase