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首页> 外文期刊>Journal of Biotechnology >Extracellular expression of Thermobifida fusca cutinase with pelB signal peptide depends on more than type II secretion pathway in Escherichia coli
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Extracellular expression of Thermobifida fusca cutinase with pelB signal peptide depends on more than type II secretion pathway in Escherichia coli

机译：带有pelB信号肽的Thermoififida fusca角质酶的细胞外表达不仅取决于大肠杆菌中的II型分泌途径

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摘要

Our previous studies demonstrated that Thermobifidafusca cutinase is released into culture medium when expressed without a signal peptide in Escherichia coli, and this extracellular expression results from an enhanced membrane permeability caused by cutinase's phospholipid hydrolase activity. The present study investigated whether this phenomenon would also occur during the expression of cutinase fused to pelB signal peptide (pe1B-cutinase). Secretion of fusion proteins of this type is generally believed to occur via type II secretion pathway. The results showed that when pe1B-cutinase was expressed in a secB knockout strain, which has a defective type II secretion pathway, there was still a large amount of cutinase in the culture medium. Additional experiments confirmed that the periplasmic and cytoplasmic fractions of the expressing cells had hydrolytic activity toward phosphatidyl ethanolamine, and the recombinant cells showed correspondingly improved membrane permeability. All these phenomena were also observed in the parent E. coli strain. Moreover, the secretion efficiency of the inactive cutinase mutant was found to be significantly lower than that of pe1B-cutinase in the parent E. co/i. Based on these results, the phospholipid hydrolase activity of pe1B-cutinase must play a larger role in its extracellular production than does type II secretion pathway. (C) 2015 Elsevier B.V. All rights reserved.
机译：我们以前的研究表明，在大肠杆菌中没有信号肽表达时，嗜热双歧角质酶会释放到培养基中，而这种细胞外表达是由角质酶的磷脂水解酶活性引起的膜通透性增强引起的。本研究调查了这种现象是否还会在与pelB信号肽（pe1B-cutinase）融合的角质酶表达过程中发生。通常认为这种类型的融合蛋白的分泌通过II型分泌途径发生。结果表明，当在具有II型分泌途径缺陷的secB敲除菌株中表达pe1B-角质酶时，培养基中仍存在大量的角质酶。另外的实验证实表达细胞的周质和细胞质级分对磷脂酰乙醇胺具有水解活性，并且重组细胞显示出相应改善的膜通透性。在亲本大肠杆菌菌株中也观察到所有这些现象。此外，发现亲本大肠杆菌中无活性角质酶突变体的分泌效率显着低于pe1B-角质酶的分泌效率。基于这些结果，与II型分泌途径相比，pe1B-角质酶的磷脂水解酶活性必须在其细胞外产生中发挥更大的作用。 （C）2015 Elsevier B.V.保留所有权利。

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