Optimization of a multi‐stage, multi‐subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites

Abstract

We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.

Protein stability: a crystallographer's perspective

“…For example, such an approach was used to remove two protease sites from a malarial vaccine candidate that was proteolytically degraded by endogenous KEX2 protease during expression. Removal of these sites enabled the production of full-length protein on a large scale (Spiegel et al, 2015). Additionally, the primary structures of some proteins are inherently unstable, with unusually short half-lives.…”

Improvement of a fermentation process for the production of two PfAMA1-DiCo-based malaria vaccine candidates in Pichia pastoris

“…Nevertheless, we previously used the three-phase strategy to express another malaria vaccine candidate (PIMP) and its variants 20 . However, we have continuously optimized the fermentation process for new vaccine candidates 21 .…”

Isolation, production and characterization of fully human monoclonal antibodies directed to Plasmodium falciparum MSP10

“…The first step of the procedure, the screening of plasma for specific antigen-binding, provides valuable information: On the one hand, the positive reactivity shows that specific antibodies are present in the subjects tested; on the other hand, it validates that the recombinant antigens produced in Nicotiana benthamiana are correctly folded. The general reactivity of these plasma samples against various P. falciparum antigens has previously been shown elsewhere [ 19 , 29 , 30 , 37 – 39 ]. Regarding the EGF-like domains of MSP10 described here, in total approx.…”

“…The definition of semi-immunity of the plasma donors was primarily based on observation that they had not shown any signs of clinical malaria for 2 years. Moreover, several screenings had been performed to make sure that the set of donors had a broad anti-malarial antibody response, as published before [ 13 , 19 , 30 , 37 – 39 ].…”