Carretas-Valdez Manuel I, Cinco-Moroyoqui Francisco J, Ezquerra-Brauer Marina J, Marquez-Rios Enrique, Quintero-Reyes Idania E, Lopez-Zavala Alonso A, Arvizu-Flores Aldo A
Departamento de Investigacion y Posgrado en Alimentos, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000, Mexico.
Departamento de Ciencias de la Salud, Campus Cajeme, Universidad de Sonora, Blvd. Bordo Nuevo s/n, Cd. Obregón, Sonora 85199, Mexico.
Protein Pept Lett. 2019;26(3):170-175. doi: 10.2174/0929866525666181019161114.
Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported.
TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium.
Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone.
The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.
鱼类来源的胰蛋白酶被认为是一种冷适应酶，可能具有潜在的生物技术应用价值。本文报道了从蒙特雷沙丁鱼（Sardinops sagax caerulea）中重组表达、复性和激活胰蛋白酶I（TryI）的过程。
TryI在大肠杆菌BL21中作为胰蛋白酶原与硫氧还蛋白的融合蛋白进行过表达。通过对细菌包涵体进行透析实现胰蛋白酶原I的复性，每升Luria肉汤培养基的回收率为16.32 mg。
在激活之前，胰蛋白酶原融合蛋白未显示出胰蛋白酶活性。通过添加从沙丁鱼幽门盲囊中纯化的0.002 U天然TryI（非重组）来激活胰蛋白酶原I。激活后的重组胰蛋白酶活性比单独的非重组胰蛋白酶高两倍。
所述方案能够从蒙特雷沙丁鱼中获得足够量的重组TryI，以进一步对其冷适应参数进行生化和生物物理表征。
