Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor
Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor
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DOI：
10.1007/s00253-004-1867-0
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发表时间：
2005-08-01
影响因子：
5
通讯作者：
Mukherjee, KJ
中科院分区：
文献类型：
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作者：
Khushoo, A;Pal, Y;Mukherjee, KJ
各种主机载体组合进行了测试，以最大限度地在大肠杆菌中的重组天冬酰胺酶的胞外生产。在BLR（DE 3）宿主细胞中在诱导型T7 lac启动子下融合至pelB前导序列的重组天冬酰胺酶的表达导致在摇瓶中的最佳胞外生产。使用该重组菌株进行分批补料研究，并使用指数补料策略以保持0.3 h（-1）的比生长速率。为了检查诱导时间对表达的影响，用1 mM异丙基-β-D-硫代吡喃半乳糖苷在不同的细胞光密度（OD 600：33，60，90，135）下诱导培养物。虽然特异性产物形成速率随着诱导OD的增加而下降，但当在OD 600为90时进行诱导时，获得了8.7 × 10（5）单位l（-1）的最大体积活性，相当于5.24 g l（-1）的重组天冬酰胺酶。重组蛋白直接从培养基中纯化，使用快速两步纯化策略，这导致回收率接近70%，比活性接近天然酶的80%。
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 (OD600: 33, 60, 90, 135). Although the specific product formation rates declined with increasing OD of induction, a maximum volumetric activity of 8.7x10(5) units l(-1), corresponding to similar to 5.24 g l(-1) of recombinant asparaginase, was obtained when induction was done at an OD600 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 similar to 70% and a specific activity of similar to 80% of that of the native enzyme.