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Refolding and Activation from Bacterial Inclusion Bodies of Trypsin I from Sardine (Sardinops sagax caerulea)

[ Vol. 26 , Issue. 3 ]

Author(s):

Manuel I. Carretas-Valdez, Francisco J. Cinco-Moroyoqui, Marina J. Ezquerra-Brauer, Enrique Marquez-Rios, Idania E. Quintero-Reyes, Alonso A. Lopez-Zavala and Aldo A. Arvizu-Flores*   Pages 170 - 175 ( 6 )

Abstract:


Background: 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.

Methods: 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.

Results: 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.

Conclusion: The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.

Keywords:

Trypsin, refolding, sardine, cold-adapted, zymogen activation, recombinant expression.

Affiliation:

Departamento de Investigacion y Posgrado en Alimentos, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000, Departamento de Investigacion y Posgrado en Alimentos, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000, Departamento de Investigacion y Posgrado en Alimentos, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000, Departamento de Investigacion y Posgrado en Alimentos, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000, Departamento de Ciencias de la Salud, Campus Cajeme, Universidad de Sonora, Blvd. Bordo Nuevo s/n, Cd. Obregón, Sonora 85199, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000, Departamento de Ciencias Quimico Biologicas, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000

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