Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield

• Published in: Biotechnology progress Vol. 28; no. 6; pp. 1419 - 1425
• Main Authors: Casteluber, Marisa C. F., Damasceno, Leonardo M., da Silveira, Wendel B., Diniz, Raphael H. S., Passos, Frederico J. V., Passos, Flávia M. L.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-11-2012; Wiley
• Subjects: aeration; Biological and medical sciences; Biomass; Bioreactors - microbiology; Biotechnology; Biotechnology - methods; Cells, Immobilized; Cloning, Molecular - methods; Fundamental and applied biological sciences. Psychology; Glycerol - metabolism; heterologous protein expression; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; recycling of biomass; streptadivin; Streptavidin - biosynthesis; Streptavidin - chemistry; Streptavidin - genetics; Ascomycota; Fungi; Yield; Pichia pastoris
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1002/btpr.1621

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