A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans
A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans
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DOI：
10.1016/j.chembiol.2018.10.017
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发表时间：
2019-02-21
影响因子：
8.6
通讯作者：
Wakarchuk, Warren
中科院分区：
文献类型：
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作者：
Du, Ting;Buenbrazo, Nakita;Wakarchuk, Warren
我们已经开发了一种用于体内生产O-糖基化蛋白的大肠杆菌菌株。这是使用双质粒方法实现的：一个编码治疗性蛋白质靶标，第二个编码O-糖基化所需的酶机制。后一种质粒编码人多肽N-乙酰半乳糖胺基转移酶以及β 1，3-半乳糖基转移酶和UDP-Glc（NAc）-4-差向异构酶，两者均来自空肠弯曲杆菌，以及细菌或人来源的二硫键异构酶。这种双质粒合成操纵子系统的有效性已经在三种具有治疗潜力的蛋白质上进行了测试：天然O-糖基化人干扰素α-2b的天然和工程化版本，以及具有一个工程化糖基化位点的人生长激素。已经建立了将核心-1聚糖添加到蛋白质上的原理证明，我们现在正在开发该系统作为在大肠杆菌中生产和修饰具有更复杂O-聚糖结构的人类蛋白质治疗剂的平台。杆菌
We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as well as a beta 1,3-galactosyl transferase and UDP-Glc (NAc)-4-epimerase, both from Campylobacter jejuni, and a disulfide bond isomerase of bacterial or human origin. The effectiveness of this two-plasmid synthetic operon system has been tested on three proteins with therapeutic potential: the native and an engineered version of the naturally O-glycosylated human interferon alpha-2b, as well as human growth hormone with one engineered site of glycosylation. Having established proof of principle for the addition of the core-1 glycan onto proteins, we are now developing this system as a platform for producing and modifying human protein therapeutics with more complex O-glycan structures in E. coli.