Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris

Abstract

Background: Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations.

Results: In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35-42%) and purity (80-85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins.

Conclusion: These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa.

Figure 1

Characterization of the growth of P. pastoris strains during the fermentation process. Time profile of optical density measurements of P. pastoris strains GS115Moz9-2, KM71HDec8-1 and X33pBaI-3 expressing recombinant Bm86, Bd86 and Ba86, respectively.

Figure 2

Characterization of protein secretion in P. pastoris strains during the fermentation process. Time profile of total protein concentration in the culture medium of P. pastoris strains GS115Moz9-2, KM71HDec8-1 and X33pBaI-3 expressing recombinant Bm86, Bd86 and Ba86, respectively.

Figure 3

Secretion of recombinant Bm86, Bd86 and Ba86 by P. pastoris. Silver stained SDS-PAGE (lanes 1–5) and Western blot analysis (lanes 6–10) of the fermentation culture supernatants after 72 hrs growing in methanol. Samples of 15 μL were loaded in each well. Membranes for Western blot were probed with serum from rabbits immunized with control Bm86 (Gavac; Revetmex) diluted 1:1000. Membranes were then washed three times with TBS and incubated with an anti-rabbit IgG HRP conjugate (Sigma-Aldrich) diluted 1:1000 in TBS. After washing the membranes again, color was developed using TMB stabilized substrate for HRP (Promega). Lanes 1 and 6: molecular weight markers (MW; ColorBurst, Sigma-Aldrich). Lanes 2 and 7: culture supernatants of the P. pastoris GS115/Albumin negative control strain. Lanes 3 and 8, 4 and 9, and 5 and 10: culture supernatants of X33pBaI-3 (Ba86), GS115Moz9-2 (Bm86) and KM71HDec8-1 (Bd86) strains, respectively. The position of recombinant proteins is indicated with arrows.

Figure 4

Characterization of purified recombinant proteins. Western blot analysis of the purified recombinant Bm86 (lane 2), Bd86 (lane 3) and Ba86 (lane 4) proteins. On each well, 3.5 μg proteins were loaded. Membranes were probed with serum from rabbits immunized with control Bm86 (Gavac; Revetmex) diluted 1:1000. Membranes were then washed three times with TBS and incubated with an anti-rabbit IgG HRP conjugate (Sigma-Aldrich) diluted 1:1000 in TBS. After washing the membranes again, color was developed using TMB stabilized substrate for HRP (Promega). Lane 1: molecular weight markers (MW; ColorBurst, Sigma-Aldrich). The position of recombinant proteins is indicated with arrows.

Figure 5

Immune cross-reactivity between Bm86 ortholog proteins. Western blot analysis of the purified recombinant Ba86 (lane 1), Bd86 (lane 2) and Bm86 (lane 3) proteins. On each well 1.5 μg proteins were loaded. Membranes were probed with serum from rabbitts immunized with recombinant Ba86 (A), Bd86 (B) and Bm86 (C) diluted 1:5000. Membranes were washed three times with TBS and incubated with an anti-rabbit IgG HRP conjugate (Sigma-Aldrich) diluted 1:1000 in TBS. After washing the membrane again, color were developed using TMB stabilized substrate for HRP (Promega). MW: molecular weight marker (ColorBurst, Sigma-Aldrich). The position of recombinant proteins is indicated with arrows.