臺灣博碩士論文加值系統

大腸桿菌表達系統被已廣泛地運用在生產異源重組蛋白質上，因為大腸桿菌的生理學與種種代謝機制已漸被研究透徹，運用上也有成熟的生物技術。不幸地，當利用大腸桿菌大量生產重組蛋白質時，往往對於細菌本身產生很大的代謝負擔反應。這種生理負擔會造成細菌的生長遲緩或抑制進而引發各種壓力反應產生負面後果，包括生產的重組蛋白質遭到蛋白脢分解、細菌生長受抑制或細胞內 rRNA 受損，其而導致核糖體瓦解和細菌死亡。在這一系列研究中，我們利用thioredoxin 和 Vitreoscilla hemoglobin (VHb) 的融合蛋白去改善在大腸桿菌生產重組蛋白質時的生理狀態，及提高細菌對於代謝負擔的抗壓性。當使用含thioredoxin 和Vitreoscilla hemoglobin (VHb) 的融合蛋白表達系統去大量生產重組蛋白質時，可以避免重組蛋白質被分解的命運且舒緩細胞產生代謝負擔，進而得到較高產量的重組蛋白質。
另外利用L-aspartate ammonia-lyase分解天冬胺酸生成丁烯二酸而提供額外的草醋酸 (OAA) 到TCA循環中，藉此改變大腸桿菌代謝的碳流量分佈，以提高中樞代謝途徑的效率，亦成功地增加重組蛋白質的產量。

Escherichia coli strain is the most commonly used producer cell for the expression of recombinant proteins. This is mainly because the biological properties of the bacterium have been well characterized, and the tools for its genetic manipulation are readily available. However, in response to a massive production of recombinant proteins, the physiological state in E. coli is greatly altered. It usually brings up the metabolic stress on the cells, usually leading to the retardation of their growth, proteolysis of recombinant proteins, disintegration of cellular rRNAs, and cell death as a result of ribosome disruption. In this study, to combat with these detrimental threats, E. coli strain was engineered to express both thioredoxin and Vitreoscilla hemoglobin for improving its physiological fitness. By a series of examination, such a strain after engineering was endowed with remarkable capabilities in protecting the produced proteins from proteolytic attack, enhancing cell growth without eliciting adverse effects caused by the metabolic burden due to the massive production of proteins in the cell, and increasing the yield of produced recombinant proteins.
Alternatively, an extra supply of intermediate metabolites in TCA cycle was approached in E. coli. This was done by the production of L-aspartate ammonia-lyase (aspartase) which mediates the conversion of aspartate to fumarate. As a result, the production of recombinant proteins could be greatly enhanced in aspartase-expressing E. coli strain.

Contents
Chapter 1. Introduction -----01
Chapter 2. Enhancement of recombinant.protein production in Escherichia coli by coproduction of aspartase -----17Chapter 3. Methods for .achieving .overproduction of recombinant proteins in stress-challenged Escherichia coli-----30
Chapter 4. Suppression of .the Clp-mediated .proteolysis in Escherichia coli by the combined act of TrxA and VHb -----55
Chapter 5. Suppression .of .the. stringent responses in Escherichia coli by the combined act of TrxA and VHb -----75

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