【作者】 郑志永；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2005， 博士

【摘要】 重组蛋白在表达过程中会耗用大量的宿主细胞资源,对大肠杆菌细胞造成代谢负荷,使细胞的生理产生很大的变化,甚至损害细胞正常的代谢功能。相应地,大肠杆菌细胞自身会针对这种变化作出应激反应来调整和适应新的生理状态。为了使大肠杆菌更好地表达重组蛋白,必须在优化目标蛋白发酵过程中考虑细胞的生理代谢。通过考察重组人表皮生长因子表达对大肠杆菌细胞生理的影响,探究重组蛋白表达与细胞生理的关系,为优化重组蛋白的发酵生产提供依据。考察了重组人表皮生长因子表达对大肠杆菌细胞生理的影响。重组蛋白的表达使大肠杆菌的生长受到抑制,降低含质粒细菌的百分比。一部分大肠杆菌向“存活但不可培养”(viable but non-culturable)生理状态分离,进一步影响重组大肠杆菌的生长。通过透射电子显微镜对诱导前后重组菌的拟核区形态进行观察,从中发现诱导后重组菌的拟核区发生了一定的松驰现象,这种松驰现象可能与染色体DNA的损伤有关,因此而影响到重组大肠杆菌的分裂能力。建立了测定细胞呼吸链脱氢酶活力的方法,并发现重组人表皮生长因子表达对细菌呼吸链脱氢酶的活力有所抑制,削弱细菌的呼吸能力。考察了胞外杂蛋白产生的原因及重组人表皮生长因子表达对胞外杂蛋白产生的影响。分析了补料分批发酵生产重组人表皮生长因子过程中大肠杆菌向“存活但不可培养”生理状态的细胞分离(cell segregation)现象,以及这种细胞分离对大肠杆菌生长和重组蛋白表达的影响。诱导剂浓度太低不利于目标重组蛋白的表达,而高诱导强度虽然能提高人表皮生长因子的初始合成速率,但也严重影响到宿主细胞的生理代谢,促使大肠杆菌向“存活但不可培养”生理状态的分离,不利于重组大肠杆菌的生长及目标蛋白的持续表达。提出了一个描述重组大肠杆菌分别向无质粒细胞分离和向“存活但不可培养”生理状态分离的动力学模型。应用该分离动力学模型对补料分批发酵生产人表皮生长因子的诱导条件进行了优化。研究了应用脉冲补料-溶氧反馈技术控制高密度培养重组大肠杆菌过程中乙酸的产生。考察了补料培养重组大肠杆菌过程中,应用脉冲补加葡萄糖时溶氧信号对乙酸产生的响应特征。当大肠杆菌以μ=0. 1 h-1的比生长速率生长时,补料过程中发酵液的乙酸含量很低,溶氧的响应信号随脉冲补料而振荡。在诱导表达重组人表皮生长因子时,重组蛋白的表达改变了细菌的生理代谢,降低细菌的最大浙江大学博士学位论文摄糖能力和摄氧能力,溶氧对脉冲补料的响应信号失去振荡特征,乙酸更容易在培养过程中积累,并反过来抑制大肠杆菌的生长和目标蛋白的持续表达。根据溶氧响应信号的特征反馈控制葡萄糖的流加速率,能有效地避免重组人表皮生长因子表达过程中乙酸的产生,实现重组大肠杆菌的高密度培养,菌体干重达到489L一‘。重组人表皮生长因子的表达水平为0.181 gL一‘,比对照组提高了45%。 研究了流加葡萄糖过程中同时流加有机氮源对大肠杆菌表达人表皮生长因子过程中细胞生理的影响,应用脉冲补料一溶氧反馈技术控制发酵过程中乙酸的产生,优化重组人表皮生长因子的发酵生产。在葡萄糖补料液中添加胰蛋白陈有助于提高培养过程中重组大肠杆菌的分裂能力,增加含质粒大肠杆菌的数量,从而降低细菌向“存活但不可培养”生理状态的分离。而提高诱导前细胞呼吸链脱氢酶的活力并同时降低诱导表达对呼吸链脱氢酶活力的削弱作用,增强细胞在诱导期间的呼吸能力,有利于大肠杆菌持续表达重组蛋白。但是,补入高浓度的胰蛋白陈不利于发酵过程中乙酸的控制,溶氧信号对脉冲补料的反馈控制的灵敏度下降,使得在脉冲补料期间乙酸容易积累,不利于重组蛋白的表达。结合过程的可操作性和培养条件对重组大肠杆菌生理代谢的影响,增强大肠杆菌在表达期间的呼吸能力及分裂能力,对表达重组人表皮生长因子的发酵过程进行了优化,细菌干重达到91 gL一,。目标蛋白产率为0.24 gL一,,比对照组提高了32.6%。关键词:大肠杆菌;细胞生理;人表皮生长因子;发酵策略更多还原

【Abstract】 The expression of recombinant protein in Escherichia coli consumes intracellular resources and exerts extra metabolic burden on the host cell. As a result, the imposition of over-weighted metabolic burden would alter and even impair the functioning and metabolism of host cell. At the same time, stress responses occur in E. coli during the expression of recombinant protein to rebalance the cell metabolism. For the sake of over-expression of target protein, cell physiological and metabolism should be considered in the optimization process.The purpose of this work is to investigate the effect of recombinant human epidermal growth factor (hEGF) expression on cell physiology in E. coli, and provide a scientific basis for optimization of recombinant protein expression.The growth inhibition of the recombinant E. coli in the expression of hEGF was observed. It was found that the recombinant cells segregated into plasmid-free cells and viable but non-culturable cells in the post-induction phase. Chromosome DNA relaxation was observed with transmitted electron microscopy during the recombinant protein expression. The DNA relaxation was proposed to correlate with impairment of chromosome DNA and could result in the decrease of dividing ability in E. coli. A colorimetric assay was carried out to measure the dehydrogenase activity of respiratory chain in E. coli, and it was found that the cell respiratory ability was inhibited in the post-induction phase of recombinant protein expression. Furthermore, the reason of extracellular proteins production and the effect of hEGF expression on extracelluar proteins production were also studied.Based on the cell ability for cell division and plasmid maintenance, the recombinant cells could be segregated into three populations: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable but non-culturable cells. The fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and recombinant protein production. The results showed that low concentration of inducer caused weak induction, whereas high levels would cause strong induction resulting in cells segregation into viable but non-culturable bacteria and low foreign protein yield. A kinetic model considering cell segregation was proposed and fitted well with the experimental data of cell growth andprotein expression. In addition, the optimal induction strategy could be predicted by the model, and was verified by the experiments with maximum expression of recombinant protein.Relations between dissolved oxygen oscillation and pulses in the glucose feed rate in fed-batch cultivation of recombinant E. coli were characterized. The dissolved oxygen response signal was used to detect the acetic acid production in pulsed fed-batch cultivation of E. coli. In fed-batch cultivation with glucose-limited growth at specific growth rate 0.1 h"1, acetic acid was kept in low level and dissolved oxygen response signal was oscillated along with the pulses in the glucose feed rate in fed-batch phase. The critical specific oxygen uptake rate and the critical specific glucose uptake rate were reduced after induction of recombinant human epidermal growth factor. Consequently, acetic acid was readily accumulated in the medium and the dissolved oxygen response signal lost its oscillation characteristics. Using feedback control of dissolved oxygen signal, the glucose feed rate in post-induction phase was effectively manipulated and acetic acid accumulation was avoided successfully. Cell concentration of 48 g L"1 dry cell weight was obtained. The level of 0.181 g L"1 human epidermal growth factor was gained and inproved 45 % comparing that in fed-batch cultivation without feedback control.The effect of tryptone addition on cell physiology in pulsed fed-batch cultivation of E. coli was investigated. Applying the feedback control of dissolved oxygen signal responding pulsed in the glucose feed rate, the acetic acid production was successfully prevented and the optimization for recombinant hEGF pro更多还原