Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3).
Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3).
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DOI：
10.1186/1475-2859-11-8
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发表时间：
2012-01-12
影响因子：
6.4
通讯作者：
Wu J
中科院分区：
文献类型：
--
作者：
Su L;Chen S;Yi L;Woodard RW;Chen J;Wu J
蛋白质的胞外表达在大规模工业化生产中具有绝对优势。我们在前期的研究中，利用大肠杆菌BL 21（DE 3）的II型分泌系统表达了主要用于纺织工业的Thermobifida fusca角质酶，并发现部分表达蛋白在周质空间中积累。由于α-溶血素分泌系统可以直接从细胞质中跨膜输出目的蛋白，因此，α-溶血素在大肠杆菌中的表达具有重要意义。因此，在本研究中，我们研究了角质酶的表达使用这种α-溶血素分泌系统。T.将fusca角质酶与α-溶血素分泌系统的特异性信号肽融合，在E. coli BL21（DE3）。此外，共表达了α-溶血素分泌系统的菌株特异性易位组分HlyB和HlyD，以促进酶的表达。对该工程细胞的培养结果表明，在相同培养条件下，培养液中角质酶活力达到334 U/ml，是II型分泌途径的2.5倍。对重组角质酶进行了纯化。纯化后的角质酶具有与野生型角质酶相似的底物特异性、pH和温度特性以及生物洗涤能力。在本研究中，T.利用α-溶血素分泌系统成功地将褐色角质酶分泌到培养基中。这是第一次报告角质酶被有效地分泌这一途径。由于利用E. coli α-溶血素分泌系统，进一步探索了该途径在工业酶中的应用。
Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system. T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase. In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.
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影响因子：
4.1
作者：
Low, Kheng Oon;Mahadi, Nor Muhammad;Illias, Rosli Md.
通讯作者：
Illias, Rosli Md.
影响因子：
3.2
作者：
Akatsuka, H;Binet, R;Omori, K
通讯作者：
Omori, K
影响因子：
4.8
作者：
Bakkes, Patrick J.;Jenewein, Stefan;Schmitt, Lutz
通讯作者：
Schmitt, Lutz
影响因子：
5.4
作者：
He Jun;Yu Bing;Chen Daiwen
通讯作者：
Chen Daiwen
影响因子：
4.7
作者：
Silva, Carla;Da, Shi;Cavaco-Paulo, Artur
通讯作者：
Cavaco-Paulo, Artur