Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine

Microbial Cell Factories

Research

Bishwanath Kumar Chourasia¹  Susheel K. Singh¹  Michael Theisen¹  Will Roeffen²  Robert W. Sauerwein²  Ulrik H. Mistarz³  Fen Yang³  Kasper D. Rand³ 

[1] Department for Congenital Disorders, Statens Serum Institut, Artillerivej 5, 2300, Copenhagen, Denmark;Department of International Health, Immunology and Microbiology, Centre for Medical Parasitology, University of Copenhagen, Copenhagen, Denmark;Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark;Department of Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands;Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark;

关键词: Plasmodium falciparum;    Malaria;    Transmission blocking;    Pf;    Lactococcus lactis;   

DOI  :  10.1186/s12934-017-0710-0

 received in 2017-02-07, accepted in 2017-05-26,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThe sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis.ResultsA series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats.ConclusionsThe established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine.

【 授权许可】

CC BY   
© The Author(s) 2017