Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli.
The C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine. Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection. F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein. F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds. F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide. Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges. On the contrary, when produced as a fusion protein with E. coli MBP, the F19 moiety was natively folded. Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast. These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo. Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein.
Planson
Anne-Gaëlle
AG
Unité de Repliement et Modélisation des Protéines, Dépt de Biochimie Structurale et Chimie, CNRS URA 2185, Institut Pasteur, 28 rue du Dr. Roux, 75015 Paris, France. planson@pasteur.fr
Guijarro
J Iñaki
JI
Goldberg
Michel E
ME
Chaffotte
Alain F
AF
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D058619:Maltose-Binding Proteins; D020066:Merozoite Surface Protein 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019897:Periplasm / Q000737:chemistry / Q000235:genetics; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
D002352:Carrier Proteins; D004220:Disulfides; D058619:Maltose-Binding Proteins; D020066:Merozoite Surface Protein 1; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins