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。
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.