Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction
Increased glycosylation efficiency of recombinant proteins in Escherichia coli by auto-induction
复制标题
通过自诱导提高大肠杆菌中重组蛋白的糖基化效率
DOI：
10.1016/j.bbrc.2017.02.037
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发表时间：
2017-03-25
影响因子：
3.1
通讯作者：
Zhang, Jianing
中科院分区：
文献类型：
--
作者：
Ding, Ning;Yang, Chunguang;Zhang, Jianing
自从大肠杆菌成功地生产N-糖基化蛋白以来，大肠杆菌被认为是生产N-糖基化蛋白的理想宿主。pgl（N-连接蛋白糖基化）基因座来自空肠弯曲杆菌。然而，利用E. COB是低效的聚糖糖基化。在这项研究中，我们开发了一种策略，生产N-糖基化的蛋白质与高效率通过优化的自动诱导方法。以人纤连蛋白III型第10结构域（FN 3）为受体蛋白模型，在其C-末端引入天然糖基化序列DFNRSK和优化的DQNAT序列，并通过柔性连接子进行改造。用抗FLAG M1抗体通过蛋白质印迹证实所得糖基化效率。将传统的IPTG诱导改为自诱导，使糖基化效率分别从60%和75%提高到90%和100%。此外，在将糖基化序列子插入FN 3环（具有局部结构构象的受体序列子）的条件下，通过我们优化的自动诱导程序，糖基化效率从35%提高到80%。为了证明优化的自动诱导方法的普遍应用的潜力，来自流感嗜血杆菌的重构lsg基因座和来自C.利用空肠，这导致100%的糖基化效率。我们的研究为优化的自诱导方法有助于在大肠杆菌中大规模生产纯的外源N-糖基化蛋白提供了定量证据。柯尔特细胞(C)2017爱思唯尔公司All rights reserved.
Escherichia colt cells have been considered as promising hosts for producing N-glycosylated proteins since the successful production of N-glycosylated protein in E. coli with the pgl (N-linked protein glycosylation) locus from Campylobacter jejuni. However, one hurdle in producing N-glycosylated proteins in large scale using E. cob is inefficient glycan glycosylation. In this study, we developed a strategy for the production of N-glycosylated proteins with high efficiency via an optimized auto-induction method. The 10th human fibronectin type III domain (FN3) was engineered with native glycosylation sequon DFNRSK and optimized DQNAT sequon in C-terminus with flexible linker as acceptor protein models. The resulting glycosylation efficiencies were confirmed by Western blots with anti-FLAG M1 antibody. Increased efficiency of glycosylation was obtained by changing the conventional IPTG induction to auto-induction method, which increased the glycosylation efficiencies from 60% and 75% up to 90% and 100% respectively. Moreover, in the condition of inserting the glycosylation sequon in the loop of FN3 (the acceptor sequon with local structural conformation), the glycosylation efficiency was increased from 35% to 80% by our optimized auto -induction procedures. To justify the potential for general application of the optimized auto -induction method, the reconstituted lsg locus from Haemophilus influenzae and PglB from C. jejuni were utilized, and this led to 100% glycosylation efficiency. Our studies provided quantitative evidence that the optimized auto-induction method will facilitate the large-scale production of pure exogenous N-glycosylation proteins in E. colt cells. (C) 2017 Elsevier Inc. All rights reserved.