Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions |
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Authors: | Georgiou George; Stephens Daren L; Stathopoulos Christos; Poetschke Heather L; Mendenhall John; Earhart Charles F |
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Affiliation: | 1Department of Chemical Engineering Austin, TX 78712, USA
3Department of Microbiology Austin, TX 78712, USA
4Division of Biological Sciences, University of Texas Austin, TX 78712, USA
5Department of Botany Austin, TX 78712, USA |
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Abstract: | Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'OmpA(46159)Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46159), a second OmpA segment, consisting ofamino acids 4666, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 4684, 46109, 46128, 46141and 46145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes. |
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Keywords: | bacterial cell-surface engineering/ Escherichia coli/ fusion protein/ ß -lactamase/ lipoprotein/ OmpA |
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