Extractive bioconversion to produce the Aedes albopictus akirin in an aqueous two-phase system supporting Pichia pastoris growth and protein secretion

Abstract

The mosquito Aedes albopictus akirin is the ortholog of the tick protective antigen, subolesin and could be used for the development of vaccines to control multiple arthropod vector species. The recombinant Pichia pastoris X33M84D8-2 strain secreting the mosquito Ae. albopictus akirin was used to design an extractive bioconversion process in an aqueous two-phase system (ATPS) to produce and purify the recombinant protein. ATPS with different compositions of polyethylene glycol 4000 (PEG4000) and 10-fold concentrated saline medium were assayed during extractive fermentation processes. The system composed of 38% PEG4000 and 12% salts supported yeast growth up to 400 O.D. 600 nm and the Ae. albopictus akirin secretion of 93 mg L −1 in the culture medium with an overall volumetric productivity of 1.03 mg L −1 h −1 and 45% purity. Both biomass and recombinant protein partitioned to the bottom phase during extractive bioconversion. After phase separation by gravity settling, a single separation process by high speed centrifugation of the bottom phase allowed cell separation yielding a recombinant protein solution with 85% purity. A conventional bioprocess was also conducted for comparison resulting in an expression level of 400 mg L −1 of the secreted protein with an overall volumetric productivity of 4.81 mg L −1 h −1 but a 33% purity at the end of the fermentation process. These results support the possibility to develop extractive bioconversion processes using ATPS for the production of mosquito akirin and other recombinant proteins in P. pastoris.