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CLONING AND EXPRESSION OF A FUNCTIONAL CORE STREPTAVIDIN IN PICHIA PASTORIS: STRATEGIES TO INCREASE YIELD
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Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunopatologia. Belo Horizonte, MG, Brazil
Universidade Federal de Viçosa. Departamento de Microbiologia. Viçosa, MG, Brazil
Universidade Federal de Viçosa. Departamento de Microbiologia. Viçosa, MG, Brazil
Universidade Federal de Viçosa. Departamento de Tecnologia de Alimentos. Viçosa, MG, Brazil
Universidade Federal de Viçosa. Departamento de Microbiologia. Viçosa, MG, Brazil
Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunopatologia. Belo Horizonte, MG, Brazil
Universidade Federal de Viçosa. Departamento de Microbiologia. Viçosa, MG, Brazil
Universidade Federal de Viçosa. Departamento de Microbiologia. Viçosa, MG, Brazil
Universidade Federal de Viçosa. Departamento de Tecnologia de Alimentos. Viçosa, MG, Brazil
Universidade Federal de Viçosa. Departamento de Microbiologia. Viçosa, MG, Brazil
Abstract
Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min(-1) , and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L(-1) ). These parameters yielded 4.0 g L(-1) of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L(-1) of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression.
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