Format

Send to

Choose Destination
Protein Expr Purif. 2011 Nov;80(1):8-16. doi: 10.1016/j.pep.2011.06.013. Epub 2011 Jul 14.

Strategies for bacterial expression of protein-peptide complexes: application to solubilization of papillomavirus E6.

Author information

1
Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, Boulevard Sébastien Brant, BP 10413, 67412 Illkirch, France.

Abstract

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif.

PMID:
21777678
PMCID:
PMC3183320
DOI:
10.1016/j.pep.2011.06.013
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center