Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Bioorg Med Chem. 2021 Mar 1;33:116037. doi: 10.1016/j.bmc.2021.116037. Epub 2021 Jan 22.
The conventional use of E. coli system for protein expression is limited to non-glycosylated proteins. While yeast, insect and mammalian systems are available to produce heterologous glycoproteins, developing an engineered E. coli-based glycosylation platform will provide a faster, more economical, and more convenient alternative. In this work, we present a two-step approach for production of a homogeneously glycosylated eukaryotic protein using the E. coli expression system. Human interferon α-2b (IFNα) is used as a model protein to illustrate this glycosylation scheme. In the first step, the N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) is co-expressed for in vivo transfer of a glucose residue to IFNα at an NX(S/T) N-glycosylation sequon. Several E. coli systems were examined to evaluate the efficiency of IFNα N-glucosylation. In the second step, the N-glucosylated protein is efficiently elaborated with biantennary sialylated complex-type N-glycan using an in vitro chemoenzymatic method. The N-glycosylated IFNα product was found to be biologically active and displayed significantly improved proteolytic stability. This work presents a feasible E. coli-based glycosylation machinery for producing therapeutic eukaryotic glycoproteins.
大肠杆菌系统常用于表达非糖基化蛋白。虽然酵母、昆虫和哺乳动物系统可用于生产异源糖蛋白，但开发基于工程大肠杆菌的糖基化平台将提供更快、更经济、更方便的替代方案。在这项工作中，我们提出了一种两步法，使用大肠杆菌表达系统生产均一糖基化的真核蛋白。人干扰素α-2b（IFNα）被用作模型蛋白来说明这种糖基化方案。在第一步中，共表达来自胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae）的 N-糖基转移酶（ApNGT），在 IFNα 的 NX（S/T）N-糖基化序列内将葡萄糖残基进行体内转移。我们考察了几种大肠杆菌系统，以评估 IFNα N-糖基化的效率。在第二步中，使用体外化学酶法有效地用双触角唾液酸化复杂型 N-聚糖对 N-糖基化蛋白进行修饰。发现 N-糖基化 IFNα 产物具有生物活性，并显示出显著提高的蛋白水解稳定性。这项工作提出了一种可行的基于大肠杆菌的糖基化机制，用于生产治疗性真核糖蛋白。
