title: Differential bacterial surface display of peptides by the transmembrane domain of OmpA.
abstract: Peptide libraries or antigenic determinants can be displayed on the surface of bacteria through insertion in a suitable outer membrane scaffold protein. Here, we inserted the well-known antibody epitopes 3xFLAG and 2xmyc in exterior loops of the transmembrane (TM) domain of OmpA. Although these highly charged epitopes were successfully displayed on the cell surface, their levels were 10-fold reduced due to degradation. We verified that the degradation was not caused by the absence of the C-terminal domain of OmpA. In contrast, a peptide that was only moderately charged (SA-1) appeared to be stably incorporated in the outer membrane at normal protein levels. Together, these results suggest that the display efficiency is sensitive to the charge of the inserted epitopes. In addition, the high-level expression of OmpA variants with surface-displayed epitopes adversely affected growth in a strain dependent, transient manner. In a MC4100 derived strain growth was affected, whereas in MC1061 derived strains growth was unaffected. Finally, results obtained using a gel-shift assay to monitor beta-barrel folding in vivo show that the insertion of small epitopes can change the heat modifiability of the OmpA TM domain from 'aberrant' to normal, and predict that some beta-barrels will not display any significant heat-modifiability at all.
authors.affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Gertjan S
authors.initials: GS
authors.lastname: Verhoeven
authors.forename: Svetlana
authors.initials: S
authors.lastname: Alexeeva
authors.forename: Marileen
authors.initials: M
authors.lastname: Dogterom
authors.forename: Tanneke
authors.initials: T
authors.lastname: den Blaauwen
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D005456:Fluorescent Dyes; D010455:Peptides; C078167:OMPA outer membrane proteins
references.citation: J Bacteriol. 2006 Jul;188(13):4627-34
references.pmid: 16788171
references.citation: Vaccine. 1990 Feb;8(1):85-91
references.pmid: 1690490
references.citation: Anal Chem. 2007 Mar 1;79(5):2174-8
references.pmid: 17253874
references.citation: PLoS Biol. 2006 Jan;4(1):e2
references.pmid: 16336047
references.citation: Trends Biotechnol. 2003 Jan;21(1):45-52
references.pmid: 12480350
references.citation: Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
references.pmid: 9092630
references.citation: Mol Microbiol. 2000 Jul;37(2):239-53
references.pmid: 10931321
references.citation: Genes Dev. 2007 Oct 15;21(20):2659-70
references.pmid: 17938245
references.citation: Mol Microbiol. 2004 Feb;51(3):645-57
references.pmid: 14731269
references.citation: J Bacteriol. 2003 Mar;185(6):2017-21
references.pmid: 12618467
references.citation: Nat Struct Mol Biol. 2006 Nov;13(11):957-64
references.pmid: 17082791
references.citation: Nat Struct Biol. 1998 Nov;5(11):1013-7
references.pmid: 9808047
references.citation: Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7457-61
references.pmid: 1502158
references.citation: Proc Natl Acad Sci U S A. 1980 Aug;77(8):4592-6
references.pmid: 7001461
references.citation: J Bacteriol. 1983 Feb;153(2):581-7
references.pmid: 6337121
references.citation: Electrophoresis. 1997 Dec;18(15):2714-23
references.pmid: 9504803
references.citation: J Mol Biol. 1986 Apr 5;188(3):491-4
references.pmid: 3525847
references.citation: Biol Chem. 2001 Dec;382(12):1669-77
references.pmid: 11843180
references.citation: J Bacteriol. 1999 Jun;181(12):3688-94
references.pmid: 10368142
references.citation: Protein Eng Des Sel. 2004 Oct;17(10):731-9
references.pmid: 15531628
references.citation: Chem Phys Lipids. 2006 Jun;141(1-2):30-47
references.pmid: 16581049
references.citation: Mol Microbiol. 2003 Jan;47(2):539-47
references.pmid: 12519203
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Sep 1;62(Pt 9):835-9
references.pmid: 16946459
references.citation: J Mol Biol. 1980 Apr;138(2):179-207
references.pmid: 6997493
references.citation: Curr Opin Struct Biol. 2007 Aug;17(4):474-80
references.pmid: 17728126
references.citation: J Biol Chem. 2003 Jul 4;278(27):24825-30
references.pmid: 12716881
references.citation: Protein Eng. 1998 Jun;11(6):489-94
references.pmid: 9725628
references.citation: J Bacteriol. 2008 Apr;190(7):2597-606
references.pmid: 18245285
references.citation: Mol Gen Genet. 1994 Apr;243(2):127-35
references.pmid: 8177210
references.citation: Gene. 1989 Oct 30;82(2):229-36
references.pmid: 2684781
references.citation: FEMS Microbiol Lett. 2007 Aug;273(1):1-11
references.pmid: 17559395
references.citation: Annu Rev Microbiol. 2007;61:191-214
references.pmid: 17506684
references.citation: Vaccine. 1994 May;12(6):492-8
references.pmid: 8036821
references.citation: Mol Microbiol. 2005 Mar;55(6):1631-45
references.pmid: 15752189
references.citation: J Cell Biol. 2003 Oct 13;163(1):35-44
references.pmid: 14530384
references.citation: FEBS Lett. 1974 May 1;41(2):195-8
references.pmid: 4604992
references.citation: Mol Divers. 1997;2(3):156-64
references.pmid: 9238646
references.citation: J Bacteriol. 1988 Apr;170(4):1533-40
references.pmid: 3280547
references.citation: Gene. 1990 Mar 30;88(1):37-45
references.pmid: 1692799
references.citation: Gene. 1987;59(1):145-50
references.pmid: 2449378