Authors: Nominé, Yves¹; Ristriani, Tutik²; Laurent, Cécile²; Lefèvre, Jean-François¹; Weiss, Étienne²

Source: Protein Engineering, Volume 14, Number 4, April 2001 , pp. 297-305(9)

Publisher: Oxford University Press

Abstract:

Recombinant production of HPV oncoprotein E6 is notoriously difficult. The unfused sequence is produced in inclusion bodies. By contrast, fusions of E6 to the C-terminus of carrier proteins such as maltose-binding protein or gluthatione-S-transferase are produced soluble. However, it has not yet been possible to purify E6 protein from such fusion constructs. Here, we show that this was due to the biophysical heterogeneity of the fusion preparations. We find that soluble MBP-E6 preparations contain two subpopulations. A major fraction is aggregated and contains exclusively misfolded E6 moieties (`soluble inclusion bodies'). A minor fraction is monodisperse and contains the properly folded E6 moieties. Using monodispersity as a screening criterion, we optimized the expression conditions, the purification process and the sequence of E6, finally obtaining stable monodisperse MBP-E6 preparations. In contrast to aggregated MBP-E6, these preparations yielded fully soluble E6 after proteolytic removal of MBP. Once purified, these E6 proteins are stable, folded and biologically active. The first biophysical measurements on pure E6 were performed. This work shows that solubility is not a sufficient criterion to check that the passenger protein in a fusion construct is properly folded and active. By contrast, monodispersity appears as a better quality criterion. The monodispersity-based strategy presented here constitutes a general method to prepare fusion proteins with optimized folding and biological activity.

Keywords: fusion protein; HPV E6 oncoprotein; monodispersity; proper folding; quality optimization

Document Type: Research article

Affiliations: 1: Laboratoire de RMN, UPR 9003 du CNRS and 2: Laboratoire d'Immunotechnologie, UPRES 1329, École Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France

Publication date: 2001-04-01

Related content

• In this: publication
• By this: publisher
• In this Subject: Chemical Engineering
• By this author: Nominé, Yves ;  Ristriani, Tutik ;  Laurent, Cécile ;  Lefèvre, Jean-François ;  Weiss, Étienne

Website © Publishing Technology. Article copyright remains with the publisher, society or author(s) as specified within the article.

• Terms and conditions
• Privacy policy
• Information for advertisers