Abstract
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.
Highlights
The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas
Since eukaryotic cells possess a sophisticated machinery for disulfide bond formation, recombinant Pfs48/45 has been produced in a range of Baculovirus (Spodoptera frugiperda Sf9) cells [9], Vaccinia virus [10], Saccharomyces cerevisiae [11], and Pichia pastoris [11], Chlamydomonas reinhardtii [12], and Nicotiana benthamiana [13]
Molecular design and expression of chimeric glutamate-rich protein (GLURP)‐Pfs48/45 fusion proteins We designed a set of fusion proteins (Fig. 1a) consisting of the Pfs48/45 6-Cys domain to address whether expression levels of secreted r0-region of the glutamate-rich protein (R0).6-Cys domain of Pfs48/45 (6C) was affected by: (1) codon optimization, (2) different signal peptides, and (3) the presence and/or position of a range of affinity-tags
Summary
The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. 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. Singh et al Microb Cell Fact (2017) 16:97 studies have identified the C-terminal portion of Pfs48/45 (6C) containing three disulfide bonds as major target of transmission blocking (TB) antibodies [4]. This Pfs48/45 region is targeted by a monoclonal antibody (mAb) mAb45.1 which promotes strong TB activity in the standard membrane feeding assay (SMFA), the gold standard for assessing transmission blockade ex vivo [5,6,7,8]. L. lactis has been used for the manufacturing of the GMZ2 malaria vaccine candidate [19,20,21,22]
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