In vitro association of fragments of a β-sheet membrane protein
Although the β-barrel membrane protein OmpA can be produced in a biologically active form in E. coli from co-expressed fragments, the fragments have not been demonstrated to associate in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex accor...
Gespeichert in:
Veröffentlicht in: | Biophysical chemistry 2010-05, Vol.148 (1), p.112-120 |
---|---|
Hauptverfasser: | , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | Although the β-barrel membrane protein OmpA can be produced in a biologically active form in
E. coli from co-expressed fragments, the fragments have not been demonstrated to associate
in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex according to the SDS band-shift assay. We are able to convert 25–35% of the fragment populations to non-covalent but SDS-stable complexes. The periplasmic chaperone Skp effectively prevented this association. Two separately expressed and purified overlapping fragments of OmpA can form a protease-resistant complex that undergoes the characteristic band-shift upon heating. Our work demonstrates that although membrane insertion and folding of β-barrel membrane proteins may be a cooperative process, the fragments can associate
in vitro without any additional components. However, the low yield and slow folding rates indicate that partially unfolded or destabilized β-sheet membrane proteins can potentially engage in many non-native interactions. |
---|---|
ISSN: | 0301-4622 1873-4200 |
DOI: | 10.1016/j.bpc.2010.03.004 |