Expression, Purification and Characterization of GMZ2’.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite

Purpose Production and characterization of a chimeric fusion protein (GMZ2’.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pf s48/45 (10C). GMZ2’.10C is a potential candidate for a mu...

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Published in:	Pharmaceutical research Vol. 34; no. 9; pp. 1970 - 1983
Main Authors:	Mistarz, Ulrik H., Singh, Susheel K., Nguyen, Tam T. T. N., Roeffen, Will, Yang, Fen, Lissau, Casper, Madsen, Søren M., Vrang, Astrid, Tiendrebeogo, Régis W., Kana, Ikhlaq H., Sauerwein, Robert W., Theisen, Michael, Rand, Kasper D.
Format:	Journal Article
Language:	English
Published:	New York Springer US 01-09-2017 Springer Springer Nature B.V
Subjects:	Amino Acid Sequence Analysis Animals Antibodies Antibody Formation Antigenic determinants Antigens Antigens, Protozoan - chemistry Antigens, Protozoan - genetics Antigens, Protozoan - immunology Antigens, Protozoan - therapeutic use Biochemistry Biomedical and Life Sciences Biomedical Engineering and Bioengineering Biomedicine C-Terminus Cloning, Molecular Cysteine Disease transmission Disulfides Enzyme-linked immunosorbent assay Fusion protein Glutamate High performance liquid chromatography Humans Lactococcus lactis Lactococcus lactis - genetics Liquid chromatography Malaria Malaria vaccine Malaria Vaccines - chemistry Malaria Vaccines - genetics Malaria Vaccines - immunology Malaria Vaccines - therapeutic use Malaria, Falciparum - immunology Malaria, Falciparum - parasitology Malaria, Falciparum - prevention & control Mass spectrometry Mass spectroscopy Medical Law Parasites Peptide mapping Pharmacology/Toxicology Pharmacy Plasmodium falciparum Plasmodium falciparum - chemistry Plasmodium falciparum - genetics Plasmodium falciparum - growth & development Plasmodium falciparum - immunology Protein binding Protein purification Protein Stability Proteins Protozoan Proteins - chemistry Protozoan Proteins - genetics Protozoan Proteins - immunology Protozoan Proteins - therapeutic use Purity Rats, Wistar Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - immunology Recombinant Fusion Proteins - therapeutic use Research Paper Rodents Sex (Psychology) Structural analysis Structural stability Tuberculosis Vaccines Vector-borne diseases Western blotting production and stability analysis of biopharmaceuticals disulphide bond mapping malaria vaccine s48/45 mass spectrometry of biopharmaceuticals Pfs48/45
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Summary:	Purpose Production and characterization of a chimeric fusion protein (GMZ2’.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pf s48/45 (10C). GMZ2’.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. Methods GMZ2’.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. Results CP-GMZ2’.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2’.10C. CP-GMZ2’.10C and IP-GMZ2’.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pf s48/45 was analysed by tandem mass spectrometry and was established for GMZ2’.10C and two reference fusion proteins encompassing similar parts of Pf s48/45. Conclusion GMZ2’.10C, combining GMZ2’ and correctly-folded Pf s48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pf s48/45 was revealed experimentally, providing an important guideline for employing the Pf s48/45 antigen in vaccine design.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0724-8741 1573-904X
DOI:	10.1007/s11095-017-2208-1