Differential bacterial surface display of peptides by the transmembrane domain of OmpA.
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.
Verhoeven
Gertjan S
GS
Molecular Cytology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands.
Alexeeva
Svetlana
S
Dogterom
Marileen
M
den Blaauwen
Tanneke
T
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*
D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D005456:Fluorescent Dyes; D010455:Peptides; C078167:OMPA outer membrane proteins
J Bacteriol. 2006 Jul;188(13):4627-34
16788171
Vaccine. 1990 Feb;8(1):85-91
1690490
Anal Chem. 2007 Mar 1;79(5):2174-8
17253874
PLoS Biol. 2006 Jan;4(1):e2
16336047
Trends Biotechnol. 2003 Jan;21(1):45-52
12480350
Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
9092630
Mol Microbiol. 2000 Jul;37(2):239-53
10931321
Genes Dev. 2007 Oct 15;21(20):2659-70
17938245
Mol Microbiol. 2004 Feb;51(3):645-57
14731269
J Bacteriol. 2003 Mar;185(6):2017-21
12618467
Nat Struct Mol Biol. 2006 Nov;13(11):957-64
17082791
Nat Struct Biol. 1998 Nov;5(11):1013-7
9808047
Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7457-61
1502158
Proc Natl Acad Sci U S A. 1980 Aug;77(8):4592-6
7001461
J Bacteriol. 1983 Feb;153(2):581-7
6337121
Electrophoresis. 1997 Dec;18(15):2714-23
9504803
J Mol Biol. 1986 Apr 5;188(3):491-4
3525847
Biol Chem. 2001 Dec;382(12):1669-77
11843180
J Bacteriol. 1999 Jun;181(12):3688-94
10368142
Protein Eng Des Sel. 2004 Oct;17(10):731-9
15531628
Chem Phys Lipids. 2006 Jun;141(1-2):30-47
16581049
Mol Microbiol. 2003 Jan;47(2):539-47
12519203
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Sep 1;62(Pt 9):835-9
16946459
J Mol Biol. 1980 Apr;138(2):179-207
6997493
Curr Opin Struct Biol. 2007 Aug;17(4):474-80
17728126
J Biol Chem. 2003 Jul 4;278(27):24825-30
12716881
Protein Eng. 1998 Jun;11(6):489-94
9725628
J Bacteriol. 2008 Apr;190(7):2597-606
18245285
Mol Gen Genet. 1994 Apr;243(2):127-35
8177210
Gene. 1989 Oct 30;82(2):229-36
2684781
FEMS Microbiol Lett. 2007 Aug;273(1):1-11
17559395
Annu Rev Microbiol. 2007;61:191-214
17506684
Vaccine. 1994 May;12(6):492-8
8036821
Mol Microbiol. 2005 Mar;55(6):1631-45
15752189
J Cell Biol. 2003 Oct 13;163(1):35-44
14530384
FEBS Lett. 1974 May 1;41(2):195-8
4604992
Mol Divers. 1997;2(3):156-64
9238646
J Bacteriol. 1988 Apr;170(4):1533-40
3280547
Gene. 1990 Mar 30;88(1):37-45
1692799
Gene. 1987;59(1):145-50
2449378