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

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, Aldo A. Arvizu-Flores*.

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 3 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


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.

[1]
Klomklao, S.; Benjakul, S.; Visessanguan, W.; Kishimura, H.; Simpson, B.K. Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol). J. Agric. Food Chem., 2006, 54(15), 5617-5622.
[2]
Arvizu-Flores, A.A.; Quintero-Reyes, I.E.; Felix-López, M.; Islas-Osuna, M.A.; Yepiz-Plascencia, G.; Pacheco-Aguilar, R.; Navare, A.; Fernández, F.M.; Velázquez-Contreras, E.F.; Sotelo-Mundo, R.R.; Castillo-Yañez, F.J. Thermodynamic activation and structural analysis of trypsin I from Monterey sardine (Sardinops sagax caerulea). Food Chem., 2012, 133, 898-904.
[3]
Siddiqui, K.S.; Cavicchioli, R. Cold-adapted enzymes. Annu. Rev. Biochem., 2006, 75, 403-433.
[4]
Feller, G.; Gerday, C. Psychrophillic enzymes: Molecular basis of cold adaptation. Cell. Mol. Life Sci., 1997, 53(10), 830-841.
[5]
Gudmundsdottir, A.; Hilmarsson, H.; Stefansson, B. Potential use of Atlantic cod trypsin in biomedicine. BioMed Res. Int., 2013, 2013, 749078.
[6]
Klomklao, S.; Benjakul, S.; Visessanguan, W.; Simpson, B.K.; Kishimura, H. Partitioning and recovery of proteinase from tuna spleen by aqueous two-phase systems. Process Biochem., 2005, 40(9), 3061-3067.
[7]
Shahidi, F.; Kamil, Y.J. Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci. Technol., 2001, 12(12), 435-464.
[8]
Macouzet, M.; Simpson, B.K.; Lee, B.H. Cloning of fish enzymes and other fish protein genes. Crit. Rev. Biotechnol., 1999, 19(3), 179-196.
[9]
Simpson, B.K.; Smith, J.P.; Haard, N.F. Encyclopedia of Food Science and Technology: Marine enzymes.In Hui, Y. H. Ed.; New York, USA, 1991, pp. 1645-1653.
[10]
Gudmundsdottir, A.; Palsdóttir, H.M. Atlantic cod trypsins: From basic research to practical applications. Mar. Biotechnol., 2005, 7(2), 77-88.
[11]
Sherry, S.; Fletcher, A.P. Proteolytic enzymes: A therapeutic evaluation. Clin. Pharmacol. Ther., 1960, 1, 202-226.
[12]
Jonsdottir, G.; Bjarnason, J.B.; Gudmundsdottir, A. Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification. Protein Expr. Purif., 2004, 33(1), 110-122.
[13]
Palsdottir, H.M.; Gudmundsdottir, A. Expression and purification of a cold-adapted group III trypsin in Escherichia coli. Protein Expr. Purif., 2007, 51(2), 243-252.
[14]
Harcum, S.W.; Bentley, W.E. Response dynamics of 26-, 34-, 39-, 54-, and 80-kDa proteases in induced cultures of recombinant Escherichia coli. Biotechnol. Bioeng., 1993, 42(6), 675-685.
[15]
Asgeirsson, B.; Fox, J.W.; Bjarnason, J.B. Purification and characterization of trypsin from the poikilotherm Gadus morhua. Eur. J. Biochem., 1989, 180(1), 85-94.
[16]
Kristjansson, M.M.; Nielsen, H.H. Purification and characterization of two chymotrypsin-like proteases from the pyloric ceca of rainbow-trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. B Biochem. Mol. Biol., 1992, 101(1-2), 247-253.
[17]
Lavallie, E.R.; Diblasio, E.A.; Kovacic, S.; Grant, K.L.; Schendel, P.F.; Mccoy, J.M. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnol., 1993, 11(2), 187-193.
[18]
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680-685.
[19]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72, 248-254.
[20]
Castillo-Yañez, F.J.; Pacheco-Aguilar, R.; Garcia-Carreno, F.L.; Navarrete-Del-Toro, M.L. Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 2005, 140(1), 91-98.
[21]
Arnorsdottir, J.; Smaradottir, R.B.; Magnusson, O.T.; Thorbjarnardottir, S.H.; Eggertsson, G.; Kristjansson, M.M. Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species. Eur. J. Biochem., 2002, 269(22), 5536-5546.
[22]
Gerday, C.; Aittaleb, M.; Bentahir, M.; Chessa, J.P.; Claverie, P.; Collins, T.; D’amico, S.; Dumont, J.; Garsoux, G.; Georlette, D.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.A.; Feller, G. Cold-adapted enzymes: From fundamentals to biotechnology. Trends Biotechnol., 2000, 18(3), 103-107.
[23]
Feller, G.; Gerday, C. Psychrophilic enzymes: Hot topics in cold adaptation. Nat. Rev. Microbiol., 2003, 1(3), 200-208.
[24]
Smalas, A.O.; Leiros, H.K.; Os, V.; Willassen, N.P. Cold adapted enzymes. Biotechnol. Annu. Rev., 2000, 6, 1-57.
[25]
Zavodszky, P.; Kardos, J.; Svingor, A.; Petsko, G.A. Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc. Natl. Acad. Sci. USA, 1998, 95(13), 7406-7411.
[26]
Sambrook, J.; Russell, D.W. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Ed.; New York, USA, 2012, pp. 1593-1599.
[27]
Hohenblum, H.; Vorauer-Uhl, K.; Katinger, H.; Mattanovich, D. Bacterial expression and refolding of human trypsinogen. J. Biotechnol., 2004, 109(1-2), 3-11.
[28]
Takahashi, S.; Ogasawara, H.; Watanabe, T.; Kumagai, M.; Inoue, H.; Hori, K. Refolding and activation of human prorenin expressed in Escherichia coli: Application of recombinant human renin for inhibitor screening. Biosci. Biotechnol. Biochem., 2006, 70(12), 2913-2918.
[29]
Kay, J.; Kassell, B. The autoactivation of trypsinogen. J. Biol. Chem., 1971, 246(21), 6661-6665.


Rights & PermissionsPrintExport Cite as


Article Details

VOLUME: 26
ISSUE: 3
Year: 2019
Page: [170 - 175]
Pages: 6
DOI: 10.2174/0929866525666181019161114
Price: $58

Article Metrics

PDF: 18
HTML: 2