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Expression of hybrid malaria antigens in insect cells and their engineering for correct folding and secretion

Published online by Cambridge University Press:  06 April 2009

V.F. Murphy ,
W.C. Rowan ,
M.J. Page  and
A.A. Holder
V.F. Murphy
Affiliation:
Department of Molecular Biology, Wellcome Biotech, Langley Court, Beckenham, Kent BR3 3BS Department of Biotechnology, Beechams Pharmaceuticals Research Division, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KTI8 5XQ.
W.C. Rowan
Affiliation:
Department of Molecular Biology, Wellcome Biotech, Langley Court, Beckenham, Kent BR3 3BS
M.J. Page
Affiliation:
Department of Molecular Biology, Wellcome Biotech, Langley Court, Beckenham, Kent BR3 3BS
A.A. Holder
Affiliation:
Department of Molecular Biology, Wellcome Biotech, Langley Court, Beckenham, Kent BR3 3BS Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA
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Summary

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 inonoclonal 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.

Keywords

baculovirus vectorscircumsporozoite proteininsect cell expressionmalaria antigens
Type
Research Article
Information
Parasitology , Volume 100 , Issue 2 , April 1990 , pp. 177 - 183
Copyright
Copyright © Cambridge University Press 1990

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