Functional cell-surface display of a lipase-specific chaperone

被引:25
作者
Wilhelm, Susanne
Rosenau, Frank
Becker, Stefan
Buest, Sebastian
Hausmann, Sascha
Kolmar, Harald
Jaeger, Karl-Erich [1 ]
机构
[1] Univ Dusseldorf, Inst Mol Enzyme Technol, Res Ctr Julich, D-52426 Julich, Germany
[2] Tech Univ Darmstadt, Dept Biochem, Clemens Schopf Inst, D-64367 Darmstadt, Germany
关键词
chaperone proteins; immunoassays; lipases; lipase-specific foldase; protein folding;
D O I
10.1002/cbic.200600203
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Liposes are important enzymes in biotechnology. Extracellular bacterial liposes 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 R 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 con be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.
引用
收藏
页码:55 / 60
页数:6
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