Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man₈GlcNAc₂ and Man₅GlcNAc₂.
Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man₈GlcNAc₂ and Man₅GlcNAc₂.
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DOI：
10.1186/1475-2859-11-53
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发表时间：
2012-05-01
影响因子：
6.4
通讯作者：
Callewaert N
中科院分区：
文献类型：
--
作者：
De Pourcq K;Vervecken W;Dewerte I;Valevska A;Van Hecke A;Callewaert N
基于蛋白质的治疗剂代表了制药行业中增长最快的一类化合物。这就产生了对强大表达系统的日益增长的需求。酵母系统被广泛使用，方便且具有成本效益。解脂耶氏酵母是通常被认为是安全的（GRAS）的合适宿主。然而，酵母菌用主要含有甘露糖的异质聚糖修饰其糖蛋白，这使下游加工复杂化，并且经常干扰人体蛋白质功能。以消除异质的酵母特异性糖基化并获得同质的人高甘露糖型糖基化。我们设计了Y在一个实施方案中，所述方法包括用解脂酶（lipolytica）处理人型末端甘露糖糖基化蛋白，以产生均质的人型末端甘露糖糖基化蛋白，即用Man 8 GlcNAc 2或Man 5GlcNAc 2糖基化的蛋白。首先，我们灭活了酵母特异性高尔基体α-1，6-甘露糖基转移酶YlOch 1 p和YlMnn 9 p;前一种失活产生了产生均一Man 8 GlcNAc 2糖蛋白的菌株。我们通过表达葡萄糖脑苷脂酶测试了该菌株，发现高甘露糖基化相关的异质性被消除。此外，对N-聚糖的详细分析表明，YlOchlp和YlMnn 9 p，尽管它们的功能最初存在一些不确定性，但最有可能是分别负责将第一个和第二个甘露糖残基添加到聚糖主链的α-1，6-甘露糖基转移酶。第二，引入ER保留的α-1，2-甘露糖苷酶产生了产生用Man 5GlcNAc 2均匀糖基化的蛋白质的菌株。内源性LIP 2 pre信号序列的使用和密码子优化极大地提高了该酶的效率。我们生成了一个Y本发明提供了用于生产用Man 8 GlcNAc 2或Man 5GlcNAc 2 N-聚糖均质糖基化的异源糖蛋白的lipolytica表达平台。该平台扩展了Y.解脂酵母作为异源表达宿主，并使生产具有均一糖基化的人高甘露糖型N-聚糖的糖蛋白成为可能，这大大拓宽了这些糖蛋白的应用范围。
Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.
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影响因子：
6.5
作者：
Alessandrini, F;Pfister, S;Behrendt, H
通讯作者：
Behrendt, H
影响因子：
11.4
作者：
NAKAYAMA, K;NAGASU, T;JIGAMI, Y
通讯作者：
JIGAMI, Y
影响因子：
3.5
作者：
Nakayama, K;NakanishiShindo, Y;Jigami, Y
通讯作者：
Jigami, Y
影响因子：
15.9
作者：
SATO, Y;BEUTLER, E
通讯作者：
BEUTLER, E
影响因子：
158.5
作者：
BARTON, NW;BRADY, RO;YU, KT
通讯作者：
YU, KT