摘要
表达外源蛋白细菌培养过程中的细胞自溶现象是一个普遍性的过程工程问题。在相同发酵培养条件下,研究生物反应器中大肠杆菌W3110表达全人源化单链抗体片段(Single chain antibody fragments,ScFv)工程菌和野生菌的外源蛋白表达及细胞活性的差异,发现外源蛋白高效表达并积累在细胞内,大部分活细胞较野生菌提前6 h进入活着但不可培养(Viable but nonculturable,VBNC)状态,进而发生细胞自溶。分析工程菌与野生菌中胁迫应激和自溶途径基因转录水平表达谱,发现外源蛋白表达过程中热激途径rpo H,dna K,dna J,gro EL,gro ES;酸胁迫途径rpoS,gadE,gadX;氧胁迫途径sodA,katE均出现表达波峰,而发酵罐中细胞自溶过程中外膜蛋白基因ompA,ompC,ompW,ompX表达量显著下降,但rpoE并未出现持续高表达。这些发现为后续ScFv表达宿主细胞抗胁迫和自溶控制策略提供了新思路。
Cell lysis is a process problem in the protein expression using bacterial host. Under the same fermentation condition,the differences in the expression o f foreign protein and cell activity between engineered and wild-type Escherichia coli W3110 expressed by the humanized single antibody fragments （ScFv） were studied. The results showed that high expression and accumulation of heterologous protein in the engineered E. coli cells were found,most living cells entered viable but nonculturable （VBNC） state 6 h in advance and the occurrence of cell lysis compared with wild- type. Thought the analysis of transcription levels of both E. coli c,we found that the process of ScFv expression causing overshoot phenomenon of multiple stressed responses pathway genes included rpoH,dnaK,dnaJ,groES,groEL in heat stress response pathway,rpoS,gadE,gadX in acid stress response patlrway, sodA ,katE in oxygen stress response patheway. However, during cell lysis process in the fennentation tank,the expression of membrane protein gene ompA ,ompC,ompW, ompX was significantly decreased,but rpoE did not appear to continue to be high expression. These findings provide us new ideas to improve the resistance of engineering bacteria cells and control cells lysis.
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2016年第9期978-986,共9页
Journal of Food Science and Biotechnology
基金
国家973计划项目(2013CB733602)
中央高校基本科研业务费专项项目(JUSRP51401A)
江苏高校优势学科建设工程项目
