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The Applied Basic Research on the Technology of Human Epidermal Growth Factor

Author: TongWangYu
Tutor: ZhuZiQiang；YaoShanZuo
School: Zhejiang University
Course: Biochemical Engineering
Keywords: Bioseparation Downstream processing Expanded bed adsorption Fermentation Gel filtration chromatography Human epidemic growth factor (hEGF) Ion exchange chromatography Isolation Kinetic model Plasmid Purification Recombinant Escherichia coli
CLC: Q819
Type: PhD thesis
Year: 2001
Downloads: 299
Quote: 3
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Abstract


The key factor is the cost of bio-separation for the high price of biotechnological products. In order to reduce the cost, the efficient ways are to improve the yield of target products, reduce the amount of byproducts and enlarge the differences of physical and chemical properties between goals and others. Therefore, the integration between fermentation and bio-separation becomes the foundation for reducing cost. According to the idea, the processing technology of human epidermal growth factor (hEGF) was reviewed in the dissertation. The influences of physical and chemical factors on the growth of recombinant Escherichia ccli, especially of operational conditions on the human epidermal growth factor (hEGF) yield and the components of cultures in fermentation were investigated. The results are below. (l)The inhibition of Ampicillin (Amp) on the cell growth was stronger than that of isopropy EJ-D-thiogalactoside (IPTG) in the recombinant Escherichia ccli. And the lag phase in the cell growth course prolonged with the decrease of inoculum volume. (2)Range analysis shows that (I) the influence of each factor on the fermentation yield of hEGF is the followings: inoculum volume> concentration of Amp > concentration of IPTG > ratio of glucose (glu)to lactose (lac) > ratio of tryptone (try) and casein (cas). (3)The optimal conditions for the fermentation of hEGF are the followings: inoculum volume of 1%, concentration of 0.3 mM IPTG, ampicillin of 150 m g l~, ratio of Lactose to glucose of 1:0 (5 g V?O g U? and ratio of tryptone to casein of 1:1 (20 g l晵 +20 g 1?. Under the conditions, hEGF concentration reached a high value of 242 mg F?and the amount of heterogeneous proteins decreased by 62% compared with that before optimization in batch fermentation. (4)The result between experimental data and the model simulation almost is consistent, especially in cell growth. Therefore, the law of hEGF fermentation can fairly be described in the established dynamics based on the analysis of fermentation system of recombinant Escherichia ccli. The influences of various operational conditions on the purification of hEGF were systematically studied on an AKTA explorer 100 using a XK 16/20 column containing STREAMLINE DEAE resin in packed bed mode. (1) The retention volume and capacity factor of hEGF increased with the increase of the linear flow rate, column bed height, pH and gradient length, but the influences of other factors on the retention volume and capacity factor were limited under the control experimental conditions. The recovery of the peak collected ascended with the decrease of the iv Abstract sample volume injected, sample concentration and gradient length as well as the increase of the column bed height and pH but the linear flow rate was in void. In addition, the width of the peak collected moved up with the increase of linear flow rate, sample volume injected, column bed height, gradient length, pH and sample concentration, but the increase of the width resulted from sample volume injected and sample concentration was very small. So the appropriate elevation of sample volume injected and sample concentration can improve the productivity of hEGF.

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