CAJ | 학술논문

刺桐胰蛋白酶抑制剂(ETI)是一种Kunitz型胰蛋白酶抑制剂,能够抑制胰蛋白酶及组织型纤溶酶原激活剂的活性,因此ETI可以作为配体与固定相偶联用于亲和层析.利用化学合成及聚合酶链式反应(PCR)的方法获得ETI的编码基因,然后用NdeⅠ/SacⅠ酶切并插入载体pET25b(+)中构建重组表达质粒pET25b(+)/ETI.重组表达质粒转化大肠杆菌BL21(DE3)后,用IPTG诱导使重组ETI(rETI)过量表达形成包涵体.包涵体经过洗涤、复性及CM离子交换柱纯化,每升培养液可获得14 mg rETI,其纯度超过92%.活性测定表明,rETI对凝血酶及尿激酶的活性没有抑制作用,但能显著抑制胰蛋白酶、瑞替普酶及纤溶酶的活性,抑制常数Ki分别为5.14×10-9,9.4×10-8和2.08×10-8mol/L.
자동이단백매억제제(ETI)시일충Kunitz형이단백매억제제,능구억제이단백매급조직형섬용매원격활제적활성,인차ETI가이작위배체여고정상우련용우친화층석.이용화학합성급취합매련식반응(PCR)적방법획득ETI적편마기인,연후용NdeⅠ/SacⅠ매절병삽입재체pET25b(+)중구건중조표체질립pET25b(+)/ETI.중조표체질립전화대장간균BL21(DE3)후,용IPTG유도사중조ETI(rETI)과량표체형성포함체.포함체경과세조、복성급CM리자교환주순화,매승배양액가획득14 mg rETI,기순도초과92%.활성측정표명,rETI대응혈매급뇨격매적활성몰유억제작용,단능현저억제이단백매、서체보매급섬용매적활성,억제상수Ki분별위5.14×10-9,9.4×10-8화2.08×10-8mol/L.
Erythrina trypsin inhibitor (ETI) is a Kunitz-type trypsin inhibitor, which inhibits both trypsin and tissue-type plasminogen activator. Immobilized ETI is an effective ligand for the affinity purification of these enzymes. In the study, the DNA encoding ETI gene was chemically synthesized and amplified with polymerase chain reaction (PCR). The amplified ETI gene was digested with NdeⅠ/SacⅠ and inserted into the pET25b ( + ) vector to construct the rETI plasmid. The recombinant expression plasmid was transformed into E. coli strain BL21 (DE3), and induced with IPTG to overexpress recombinant ETI as insoluble inclusion body. After inclusion body purification, renaturation and CM cation-exchange chromatography, 14 mg rETI were obtained from one liter of culture medium (3.5 g pellet mass) with homogeneity greater than 92%. Kinetic analysis showed that rETI had no inhibitory effect on thrombin and urokinase, but significantly inhibit the activity of trypsin, reteplase and plasmin with a Ki of 5.14 × 10-9, 9.4 × 10-8 and 2.08 × 10-8 mol/L respectively.