不同培養基與誘導劑濃度對基因重組半胱氨酸蛋白酶抑制劑之活性表現的影響

The effect of various media and inducer concentration on the expression of recombinant cystatin

謝政翰(Cheng-Han Hsieh)

指導教授 ： 陳志成

大同大學/工程學院/生物工程學系所/碩士(2005年)

摘要

芋頭中的半胱氨酸蛋白酶抑制劑（Cystatin）具有抗線蟲或鱗翅目昆蟲之功能。本研究以基因重組Escherichia coli XL1-blue/pGEX-CeCPI於搖瓶培養中生產半胱氨酸蛋白酶抑制劑（Cystatin）。探討Cystatin生產時，應根據何種原則添加誘導劑Isopropyl-β-D-thiogalactopyranoside (IPTG)。因尚無文獻報導 Cystatin 之抑制活性的簡易測量方法，而本實驗則發展了一種測量 Cystatin 抑制比活性的簡易方法。在實驗中發現，添加0.2 mM IPTG進行誘導時，不論以Luria Broth （LB） 或是Expansion Broth/Overexpression Broth （EB/OB）為培養基，Cystatin 的產量能夠於12小時達到最高，以EB/OB為培養基生產之Cystatin 抑制比活性最高值是以 LB 為培養基的1.33倍。若以0.2 mM IPTG 進行誘導，以有無加入甘油當抗凍劑進行保存破菌後的蛋白質萃取液做比較時，當未加入甘油，以EB/OB為培養基生產之 Cystatin 抑制比活性最高值是以LB為培養基的3.58倍。若保存時加入甘油，以LB為培養基生產之 Cystatin 抑制比活性，其最高值是未加甘油組的1.55倍，以 EB/OB 為培養基生產之 Cystatin 抑制比活性，其最高值是未加甘油組的1.47倍。

關鍵字

抑制比活性 ； 誘導劑 ； 半胱氨酸蛋白酶抑制劑

並列摘要

This research is base on Escherichia coli XL1-blue/pGEX-CeCPI in flask experiment to generate Cystatin。What principles should we follow to add Isopropyl-β-D-thiogalactopyranoside (IPTG) When Cystatin generates. Due to there is no assay reveals the specific activity of Cystatin. Moreover, during this experiment, we could define Cystatin as Cystatin Unit. Not only by adding Cystatin reactive volume（g）can be described. In this experiment, we learn that while we add 0.2 m IPTG to induce. No matter we use Luria Broth（LB）or Expansion Broth / Overexpression（EB/OB） Broth as the medium, the capacity of Cystatin can both reach the highest within 12 hours. But if we use EB/OB as the medium to generate Cystatin, the specific activity is 1.33 times to the one to use LB as a medium.。
Given that induced by 0.2 mM IPTG and stored by glycerol as a reagent of freeze resistant , the specific activity will reach its highest after induced by LB or EB/OB as a medium along with glycerol excluded. Cystatin specific activity produced by EB/OB as a medium is 3.58 times than that by LB as a medium. Should glycerol is included, the specific activity will reach its highest at 3 hours after induced by LB or EB/OB as a medium. The cystatin specific activity produced by LB as a medium is 1.58 times than that without glycerol, while by EB/OB as a medium is 1.47 times that without glycerol.

並列關鍵字

CeCPI ； cystatin ； protease ； specific activity

參考文獻

楊藹華，2004，芋頭半胱胺酸蛋白酶抑制劑(Tarocystatin)之基因選殖、表現特性及對微生物抗性的分子機制研究，國立台灣大學植物科學研究所博士論文。

楊藹華，2000，漫談生物技術在作物抗蟲之應用，台南區農業專訊第32期。

Abe, K., Y. Emori, H. Kondo, K. Suzuki. and S.Arai. 1987. Purification and properties of a cysteine proteinase from germinating rice seeds. Agric. Biol. Chem. 51(6):1509-1514.

Abe, M. K. Abe., M. Kurodo. and S.Arai. 1992. Corn kernel cysteine proteinase inhibitor as a novel superfamily member of plant origin: molecular cloning and expression studies. Eur. J. Biochem. 209:933-937.

Amann, E. Brosius, J., Ptashne, M. 1983, Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli gene. 25：167-168.