Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor.
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.
Khushoo
Amardeep
A
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Pal
Yogender
Y
Mukherjee
K J
KJ
D001215:Asparaginase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019149:Bioreactors / Q000382:microbiology*; D017077:Culture Media, Conditioned / Q000378:metabolism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000031:analogs & derivatives / Q000494:pharmacology; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; C070641:isopropyl-beta-galactopyranoside; D001215:Asparaginase