High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins.
High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins.
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DOI：
10.1371/journal.pone.0120458
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Zhai C
中科院分区：
文献类型：
--
作者：
Wang F;Wang X;Yu X;Fu L;Liu Y;Ma L;Zhai C
内切-β-N-乙酰氨基葡萄糖苷酶H(Endo H，EC3.2.1.96)是一种广泛应用于糖蛋白研究的糖水解酶。本研究旨在评价内切β-N-乙酰氨基葡萄糖苷酶H在巴斯德毕赤酵母中高效表达的效果。根据毕赤酵母密码子使用的偏好性，优化了该酶的DNA编码序列，并通过重叠聚合酶链式反应合成了该酶的DNA编码序列。将该新基因克隆到pHBM905A载体中，并导入巴斯德毕赤酵母GS115中进行分泌表达。在摇瓶中用1%(v/v)的甲醇诱导6d后，目的蛋白的产量约为397 mg/L，远高于在大肠杆菌和家蚕中异源表达的产量。对重组酶进行了纯化，并对其酶学性质进行了研究。其比活力为461573 U/mg。其最适pH为5.5，最适温度为37℃。此外，我们的研究表明，通过该重组菌与表达底物的菌株共发酵或将内切-β-N-乙酰氨基葡萄糖苷酶H表达菌株的培养上清液与表达底物的菌株发酵后混合，可以有效地去除在毕赤酵母中表达的几种重组蛋白的N-连接糖链。本文首次报道了内切β-N-乙酰氨基葡萄糖苷酶H在巴斯德毕赤酵母中的高效表达及其在异源糖蛋白脱糖反应中的应用。
Endo-β-N-acetylglucosaminidase H (Endo H, EC3.2.1.96) is a glycohydrolase that is widely used in the study of glycoproteins. The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris. The DNA coding sequence of this enzyme was optimized based on the codon usage bias of Pichia pastoris and synthesized through overlapping PCR. This novel gene was cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretary expression. The yield of the target protein reached approximately 397 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks, which is much higher than that observed upon heterologous expression in Escherichia coli and silkworm. This recombinant enzyme was purified and its enzymatic features were studied. Its specific activity was 461573 U/mg. Its optimum pH and temperature were pH 5.5 and 37°C, respectively. Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-β-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation. This is the first report of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.
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