Various host-vector combinations were tested to maximize the extracellular production of recombinant asparaginase in Escherichia coli. Expression of recombinant asparaginase fused to pelB leader sequence under the inducible T7lac promoter in BLR (DE3) host cells resulted in optimum extracellular production in shake-flasks. Fed-batch studies were carried out using this recombinant strain and an exponential feeding strategy was used to maintain a specific growth rate of 0.3 h(-1). To check the effect of the time of induction on expression, cultures were induced with 1 mM isopropyl-beta-D-thiogalactopyranoside at varying cell optical densities (OD(600): 33, 60, 90, 135). Although the specific product formation rates declined with increasing OD of induction, a maximum volumetric activity of 8.7 x 10(5) units l(-1), corresponding to approximately 5.24 g l(-1) of recombinant asparaginase, was obtained when induction was done at an OD(600) of 90. The recombinant protein was purified directly from the culture medium, using a rapid two-step purification strategy, which resulted in a recovery of approximately 70% and a specific activity of approximately 80% of that of the native enzyme.
Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal alpha peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h(-1) for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y (P/X)) was approximately 2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l(-1) h(-1).
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