A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein.
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 glutathione-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.
Nominé
Y
Y
Laboratoire de RMN, UPR 9003 du CNRS and Laboratoire d'Immunotechnologie, UPRES 1329, Ecole Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France.
Ristriani
T
T
Laurent
C
C
Lefèvre
J F
JF
Weiss E
Travé
G
G
D002352:Carrier Proteins / Q000494:pharmacology; D019281:Dimerization; D005982:Glutathione Transferase / Q000494:pharmacology; D006801:Humans; D007181:Inclusion Bodies, Viral / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009856:Oncogene Proteins, Viral / Q000737:chemistry*; D027383:Papillomaviridae / Q000737:chemistry; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D012097:Repressor Proteins; D012995:Solubility; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification
D002352:Carrier Proteins; C068651:E6 protein, Human papillomavirus type 16; D058619:Maltose-Binding Proteins; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014760:Viral Fusion Proteins; D005982:Glutathione Transferase