Expression of a Beauveria bassiana chitinase (Bbchit1) in Escherichia coli and Pichia pastoris
Expression of a Beauveria bassiana chitinase (Bbchit1) in Escherichia coli and Pichia pastoris
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白僵菌几丁质酶 (Bbchit1) 在大肠杆菌和毕赤酵母中的表达
DOI：
10.1016/j.pep.2007.06.012
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发表时间：
2007-11-01
影响因子：
1.6
通讯作者：
Pei, Yan
中科院分区：
文献类型：
--
作者：
Fan, Yanhua;Zhang, Yongjun;Pei, Yan
球孢白僵菌几丁质酶（Beauveria bassiana chitinase，Bbchitl）是一种重要的角质层降解酶，参与真菌对昆虫的致病作用。为了获得足够活性的几丁质酶用于体外功能分析，分别在大肠杆菌和毕赤酵母中表达了Bbehitl基因。重组Bbchitl在大肠杆菌表达系统中得到了高效表达，但主要以包涵体形式存在。通过透析实现溶解的包涵体蛋白的重折叠。在毕赤酵母表达系统中，Bbchitl在甲醇诱导下分泌到培养基中。通过柱层析和阴离子交换层析从培养基中纯化活性Bbchitl至接近90%的纯度。毕赤酵母表达的Bbchit I的产量为153 mg/L，显著高于大肠杆菌包涵体复性的Bbchit I的产量（50 mg/L）。此外，毕赤酵母Bbchit]的比活也高于E.大肠杆菌（3.9 U/mg对2.8 U/mg）。这些结果表明，毕赤酵母是一种高效表达Bbehit]的简便表达系统。(c)2007年爱思唯尔公司好的reserved.
Beauveria bassiana chitinase (Bbchitl) is an important cuticle degrading enzyme involved in pathogenesis of fungi against insect. To obtain enough active chitinase for performing in vitro functional analysis, Bbehitl gene was expressed in Escherichia coli and Pichiapastoris, respectively. The high-level production of recombinant Bbchitl was detected in E coli expression system, however mainly located in inclusion bodies. Refolding of solubilized inclusion body proteins was achieved by dialysis. In P. pastoris expression system, Bbchitl was secreted into the culture medium under the induction of methanol. Active Bbchitl was purified to near 90% purity from culture medium by desalting chromatography and anion exchange chromatography. The yield of Bbchit I produced by P. pastoris was estimated at 153 mg/L, significantly higher than that of the refolded Bbchitl from E coli inclusion bodies (50 mg/L). Additionally, the specific activity of Bbchit] from P. pastoris was also higher than that from E. coli (3.9 U/mg versus 2.8 U/mg). These results indicated P. pastoris was a convenient expression system for the efficient production of Bbehit]. (c) 2007 Elsevier Inc. All rights. reserved.