Improving intracellular production of recombinant protein in Pichia pastoris using an optimized preinduction glycerol-feeding scheme.

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l(-1), short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l(-1). Thus, a novel strategy of induction at a cell density of 320 g l(-1) was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h(-1). The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g(-1) h(-1) from 0.043 mg g(-1) h(-1). Consequently, both high cell density (428 g l(-1)) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h(-1) to the cell density of 320 g l(-1) and inducing the expression by mixed feeding.