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Pharm Res. 2017 Sep;34(9):1970-1983. doi: 10.1007/s11095-017-2208-1. Epub 2017 Jun 23.

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.

Author information

1
Department of Pharmacy, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
2
Department for Congenital Disorders, Statens Serum Institut, Artillerivej 5, Copenhagen, Denmark.
3
Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark.
4
Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
5
Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
6
Bioneer A/S, Hørsholm, Denmark.
7
Department for Congenital Disorders, Statens Serum Institut, Artillerivej 5, Copenhagen, Denmark. mth@ssi.dk.
8
Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark. mth@ssi.dk.
9
Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. mth@ssi.dk.
10
Department of Pharmacy, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark. kasper.rand@sund.ku.dk.

Abstract

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 Pfs48/45 (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 Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45.

CONCLUSION:

GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 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 Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.

KEYWORDS:

Pfs48/45; disulphide bond mapping; malaria vaccine; mass spectrometry of biopharmaceuticals; production and stability analysis of biopharmaceuticals

PMID:
28646324
DOI:
10.1007/s11095-017-2208-1
[Indexed for MEDLINE]

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