吳郭魚類胰島素生長因子第二型在大腸桿菌及飼料酵母菌之表現與生產條件探討

Studies on the Expression and Production Conditions of Tilapia Insulin-Like Growth Factor-2 in Escherichia coli and Candida utilis

胡紹揚(Shao-Yang Hu)

指導教授 ： 黃健雄

共同指導教授 ： 吳金洌(Jen-Leih Wu)

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.10090

摘要

完整成熟之吳郭魚類胰島素生長因子第二型(IGF-2)利用載體pET28b(+)於大腸桿菌中表現，三角瓶中帶有pET-IGF2重組質體之E. coli BL21(DE3)於培養第3小時以0.06 mM IPTG 誘導可獲得最高表現量佔菌體總蛋白之10.3%，主要以內涵體之形式存在。為了大量生產含有重組IGF-2之菌體，利用發酵槽以pH-stat控制原理進行高細胞密度培養，在不誘導情況下，經30小時培養後可獲得菌體濃度OD 600nm 與菌體乾重分別為450與183 g l-1 DCW，產率為6.1 g l-1 DCW h-1，培養最終菌體仍保有91％之表現質體，在培養第18小時 (114.5 g l-1 DCW )進行添加0.06 mM IPTG誘導，可於培養之第30小時可獲得菌體乾重156 g l-1 DCW，其表現量佔菌體總蛋白11.3％，相當於每公升發酵液可獲得9.69 g之IGF-2。利用簡單內涵體回收純化步驟，可由22.5 ml發酵液獲得19.51 mg 純度為90% 之IGF-2，回收率為20.5%，相當於每公升發酵液可獲得869.67 mg純化之IGF-2，純化之IGF-2其N端序列分析結果與原態IGF-2序列相同，質譜分析分子量為9072.8 Da。 MTT呈色結果顯示純化之IGF-2對ZFL、TO-2、MRC-5與Balb/3T3 clone 31A四種細胞株具有促進生長活性，以Balb/3T3 clone 31A 細胞株效果最好，利用此活性分析方法定義出生物活性單位，可有效評估菌體生物活性含量，方便於菌體飼料生產加工與儲藏安定之應用。飼料酵母菌Candida utilis首先做為IGF-2之表現宿主，利用糖蜜培養基可有效誘導IGF-2之表現，表現量佔可溶性蛋白6.2%，每克酵母菌體IGF-2含量為7.17 mg，純化之IGF-2以吳郭魚類胰島素生長因子抗體確認後，質譜分析測定其分子量 10717.9 Da，顯示無醣化作用。當添加濃度為120 nM時，可促進Balb/3T3 Clone31A細胞株達生長促進效果74.18±10.73%，相當於純化之酵母IGF-2含有生物活性單位4.73±0.50 x 103 U mg-1，利用糖蜜做為IGF-2酵母表現菌株生產原料，三角瓶培養顯示添加60 g l-1 corn steep liquor (CSL)對菌體生長有最佳提升效果，五公升發酵槽中以固定速率饋料模式進行培養，饋料液中分別添加葡萄糖與CSL作為碳源及生長因子補充，培養84小時後可獲得菌體濃度與菌體乾重分別為OD 600nm 657.52與177.58 g l-1 DCW，產率達2.114 g l-1 DCW h-1，對糖收率為0.423 g g-1，有效完成最大菌體濃度之高細胞密度培養目標。分析酵母菌體之IGF-2表現量佔可溶性蛋白質5.2%，每克菌體乾重含有IGF-2 6.23 mg，相當於每克菌體含有生物活性單位2.95 x 104 U。

關鍵字

飼料酵母菌 ； 大腸桿菌 ； 類胰島素生長因子

並列摘要

The mature tilapia insulin-like growth-2 (IGF-2) cDNA was cloned into pET28b(+) vector and expressed in Escherichia coli. In flask culture, the transformant E. coli BL21(DE3) harboring pET-IGF2 expressed the recombinant IGF-2 as inclusion bodies, and the content was as high as 10.3% of the total protein content after 9 hr 0.06 mM IPTG induction. For production of recombinant IGF-2 in E. coli, pH-stat fed-batch cultures were used to achieve a high cell density culture. A cell concentration OD 183 g l-1 dry cell weight and productivity 6.1 g l-1 DCW h-1 were obtained after 30 h cultivation and plasmid stability was maintained at 91% levels. When cells were induced at a cell concentration of 114.5 g l-1 dry cell weight, the biomass was 156 g l-1 dry cell weight and the amount of IGF-2 produced was 9.69 g l-1 (11.3% of the total protein). Using a simple purification process, 19.51 mg of insulin-like growth factor-2 was obtained from a 22.5-ml of culture, and the purity and the recovery yield were 90% and 20.5%, respectively, that equal to 869.67 mg of IGF-2 could obtained from one liter of cluture. The N-terminal amino acid sequencing of purified IGF-2 was identical to that of the native IGF-2 and the mass spectrum analysis of purified IGF-2 was 9072.8 Da. The biological activity of the purified IGF-2 was demonstrated as promoting the growth of four different cell lines, ZFL, TO-2, MRC-5 and Balb/3T3 clone 31A by the colorimetric bioassay and the best growth stimulation ratio was obtained for the Balb/3T3 clone 31A cell line. Using the bioassay method, the unit of bioactivity could be defined to evaluate the content of bioactivity in the cell, and that is convenient in application of cell production and stability of storage. Candida utilis was first time used as host for expression of IGF-2 and IGF-2 could be produced by using molasses medium as an inducer. The IGF-2 content was as high as 6.2 % of soluble protein and 7.17 mg of IGF-2 per gram of cell in the flask cultures. Purified IGF-2 was identified by anti-mouse IGF-2 antibody and the molecular mass was 10717.9 Da by the mass spectrum analysis, showing that there is no glycosylation with it. A growth stimulation ratio of 74.18±10.73 % was obtained in the presence of 120 nM of purified IGF-2，that equal to 4.73±0.50 x 103 U mg-1 of bioactivity. Molasses was used as material to produce yeast cell due to the lower cost. The best improve effect on cell growth when 60 g l-1 of corn steep liquor was added in the molasses medium in the flask cultures. In 5 l fermentor, fed-batch culture was used to high-level produce yeast cell with constant feeding. Glucose and CSL were used to supply carbon source and growth factor in feeding solution, respectively. After 84 h cultivation, the cell concentration and biomass were OD 600nm 657.52 and 177.58 g l-1 DCW, respectively. The productivity was reached to 2.114 g l-1 DCW h-1 and the yield coefficient for glucose was 0.423 g g-1. A high cell density cultivation which closes to the maximum cell concentration was accomplished. The IGF-2 content was 5.2% of soluble protein and 6.23 mg in one gram yeast cell. This result indicated that equal to 2.95 x 104 U of bioactivity in one gram of yeast cell.

並列關鍵字

Escherichia coli ； Insulin-Like Growth Factor-2 ； Candida utilis

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