生物反应器中表达ScFv大肠杆菌细胞自溶的分析

doi:10.3979//j.issn.1673-1689.2016.09.012

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生物反应器中表达ScFv大肠杆菌细胞自溶的分析
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                        10.3979//j.issn.1673-1689.2016.09.012
                    
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Cell Lysis Analysis of E.coli Expressing ScFv in Bioreactor

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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rpoH，dnaK，dnaJ，groEL，groES；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。

Abstract:

Cell lysis is a process problem in the protein expression using bacterial host. Under the same fermentation condition，the differences in the expression of 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 pathway，sodA，katE in oxygen stress response patheway. However，during cell lysis process in the fermentation 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.

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胡卫国,安展飞,刘秀霞,戴晓峰,杨艳坤,白仲虎.生物反应器中表达ScFv大肠杆菌细胞自溶的分析[J].食品与生物技术学报,2016,35(9):978-986.

HU Weiguo, AN Zhanfei, LIU Xiuxia, DAI Xiaofeng, YANG Yankun, BAI Zhonghu. Cell Lysis Analysis of E. coli Expressing ScFv in Bioreactor[J]. Journal of Food Science and Biotechnology,2016,35(9):978-986.

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在线发布日期: 2016-11-01
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