title: Automated purification of recombinant proteins: combining high-throughput with high yield.
abstract: Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli (E. coli). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni-NTA-Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 microg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 microg to 45 mg per purification run, as well as strategies for optimizing these protocols.
authors.affiliation: Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Chiann-Tso
authors.initials: CT
authors.lastname: Lin
authors.forename: Priscilla A
authors.initials: PA
authors.lastname: Moore
authors.forename: Deanna L
authors.initials: DL
authors.lastname: Auberry
authors.forename: Elizabeth V
authors.initials: EV
authors.lastname: Landorf
authors.forename: Terese
authors.initials: T
authors.lastname: Peppler
authors.forename: Kristin D
authors.initials: KD
authors.lastname: Victry
authors.forename: Frank R
authors.initials: FR
authors.lastname: Collart
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kery
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002474:Cell-Free System; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D005374:Filtration; D008280:Magnetics; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D020592:Shewanella / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D013010:Sonication
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins