Department of Chemical and Biological Engineering, ChELSI Institute, Biological and Environmental Systems Group, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK.
Biotechnol Bioeng. 2011 Apr;108(4):902-12. doi: 10.1002/bit.23011. Epub 2010 Dec 22.
Recently, the prospect of using Escherichia coli as a host for human glycoprotein production has increased due to detailed characterization of the prokaryotic N-glycosylation process and the ability to transfer the system into this bacterium. Although functionality of the native Campylobacter jejuni N-glycosylation system in E. coli has been demonstrated, the efficiency of the process using the well-characterized C. jejuni glycoprotein AcrA, was found to be low at 13.4±0.9% of total extracted protein. A combined approach using isobaric labeling of peptides and probability-based network analysis of metabolic changes was applied to forward engineer E. coli to improve glycosylation efficiency of AcrA. Enhancing flux through the glyoxylate cycle was identified as a potential metabolic manipulation to improve modification efficiency and was achieved by increasing the expression of isocitrate lyase. While the overall recombinant protein titre did not change significantly, the amount of glycosylated protein increased by approximately 300%.
最近，由于对原核 N-糖基化过程的详细表征以及将该系统转移到这种细菌中的能力，使用大肠杆菌作为人糖蛋白生产宿主的前景有所增加。尽管已经证明了在大肠杆菌中使用天然空肠弯曲菌 N-糖基化系统的功能，但使用经过充分表征的空肠弯曲菌糖蛋白 AcrA 的过程效率较低，仅为总提取蛋白的 13.4±0.9%。采用肽的等压标记和基于概率的代谢变化网络分析的联合方法，正向工程大肠杆菌以提高 AcrA 的糖基化效率。通过增加异柠檬酸裂解酶的表达，鉴定出增强乙醛酸循环通量是提高修饰效率的一种潜在代谢操作。虽然整体重组蛋白产量没有显著变化，但糖基化蛋白的量增加了约 300%。
