title: Functional cell-surface display of a lipase-specific chaperone.
abstract: Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Research Centre Jülich, 52426 Jülich, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Wilhelm
authors.forename: Frank
authors.initials: F
authors.lastname: Rosenau
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.forename: Sebastian
authors.initials: S
authors.lastname: Buest
authors.forename: Sascha
authors.initials: S
authors.lastname: Hausmann
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
mesh_terms: D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002469:Cell Separation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D015723:Gene Library; D008049:Lipase / Q000737:chemistry*; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011549:Pseudomonas / Q000378:metabolism
chemical_list: D018832:Molecular Chaperones; D008049:Lipase