Expression of hybrid malaria antigens in insect cells and their engineering for correct folding and secretion.
Hybrid proteins containing selected regions of the major surface antigens of the sporozoite and merozoite stages of Plasmodium falciparum were expressed in insect cells using baculovirus vectors. A recombinant protein containing the signal peptide from the precursor to the major merozoite surface antigens (PMMSA) fused to a fragment from the carboxy (C) terminus of the same gene was recognized by monoclonal antibodies specific for reduction-sensitive conformational epitopes within the C-terminal fragment, suggesting that correct disulphide cross-linking of cysteine residues within this region had occurred. Addition of 26 copies of the tetrapeptide repeat from the circumsporozoite protein (CSP) resulted in a protein recognized by anti-CSP antiserum as well as the conformation specific monoclonal antibodies. Deletion of the C-terminal putative anchor sequence from both proteins resulted in secretion of protein in a fully soluble form antigenically indistinguishable from the anchor containing products. Correct conformation was not observed when the proteins were expressed as polyhedrin fusions without the signal peptide. These data indicate that the PMMSA signal peptide is recognized in insect cells and that correct assembly of disulphide cross-links is dependent upon targeting the protein to the endoplasmic reticulum.
Murphy
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VF
Department of Molecular Biology, Wellcome Biotech, Langley Court, Beckenham, Kent.
Rowan
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WC
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MJ
Holder
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D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths; D010957:Plasmids; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology*; D015183:Restriction Mapping
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D004247:DNA