Cloning and expression of a functional core streptavidin in Pichia pastoris: strategies to increase yield.
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.
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Marisa C F
MC
Casteluber
Leonardo M
LM
Damasceno
Wendel B
WB
da Silveira
Raphael H S
RH
Diniz
Frederico J V
FJ
Passos
Flávia M L
FM
Passos
D018533:Biomass; D019149:Bioreactors / Q000382:microbiology*; D001709:Biotechnology / Q000379:methods*; D018914:Cells, Immobilized; D003001:Cloning, Molecular / Q000379:methods; D005990:Glycerol / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019809:Streptavidin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D019809:Streptavidin; D005990:Glycerol