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Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield
Author(s) -
Casteluber Marisa C. F.,
Damasceno Leonardo M.,
da Silveira Wendel B.,
Diniz Raphael H. S.,
Passos Frederico J. V.,
Passos Flávia M. L.
Publication year - 2012
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.1621
Subject(s) - pichia pastoris , bioreactor , biomass (ecology) , streptavidin , glycerol , aeration , chemistry , recombinant dna , biotinylation , heterologous , yield (engineering) , chromatography , food science , biochemistry , biotin , biology , materials science , gene , organic chemistry , agronomy , metallurgy
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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012