Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.
Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.
复制标题
用于提高大肠杆菌糖基化效率的系统代谢工程。
DOI：
10.1016/j.bbrc.2012.02.020
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发表时间：
2012-03-16
影响因子：
3.1
通讯作者：
Wright PC
中科院分区：
文献类型：
--
作者：
Pandhal J;Desai P;Walpole C;Doroudi L;Malyshev D;Wright PC
大肠杆菌细胞需要改造，使其成为生产糖蛋白的理想宿主。►密码子优化pglB可提高糖基化效率。脂质连接的前体似乎并不限制E。coli N-糖基化。增加WecA的表达可以提高E. coli N-糖基化效率。最近，增加大肠杆菌细胞在工业应用中具有的用于重组蛋白生产的工具包的努力已经导致了制备糖基化治疗性蛋白的稳步进展。尽管制备具有精细的人型聚糖的治疗相关的复杂蛋白质的期望是一个主要目标，但在大肠杆菌中外源蛋白质的N-糖基化过程的相对较差的效率。大肠杆菌仍然是工业采用的障碍。在这项研究中，一个系统的方法被用来增加糖蛋白生产滴度的一个范例蛋白质，AcrA，并使用蛋白质印迹和伪选择性反应监测（pSRM）的组合量化所得的糖基化效率。Western blot和pSRM结果表明，密码子优化的寡糖基转移酶，PglB的E。大肠杆菌表达，效率分别提高了77%和101%。此外，增加糖基转移酶WecA在E. coli的效率分别提高了43%和27%。然而，增加糖基化过程中使用的供体脂质的量不会影响该系统中使用该特定蛋白质的糖基化效率。
► Escherichia coli cells require modifying to make ideal hosts for producing glycoproteins. ► Codon optimising pglB leads to increased glycosylation efficiency. ► Lipid linked precursors do not appear to be limiting for E. coli N-glycosylation. ► Increasing expression of WecA improves E. coli N-glycosylation efficiency. Recently, efforts to increase the toolkit which Escherichia coli cells possess for recombinant protein production in industrial applications, has led to steady progress towards making glycosylated therapeutic proteins. Although the desire to make therapeutically relevant complex proteins with elaborate human-type glycans is a major goal, the relatively poor efficiency of the N-glycosylation process of foreign proteins in E. coli remains a hindrance for industry take-up. In this study, a systematic approach was used to increase glycoprotein production titres of an exemplar protein, AcrA, and the resulting glycosylation efficiency was quantified using a combination of Western blots and pseudo Selective Reaction Monitoring (pSRM). Western blot and pSRM results demonstrate that codon optimising the oligosaccharyltransferase, PglB, for E. coli expression, increases efficiency by 77% and 101%, respectively. Furthermore, increasing expression of glycosyltransferase, WecA, in E. coli improves efficiency by 43% and 27%, respectively. However, increasing the amount of donor lipid used in the glycosylation process did not impact on the glycosylation efficiency in this system, with this specific protein.