Abstract
Lipoxygenases are non-heme iron-containing lipid dioxygenases enzymes that catalyze the hydroperoxidation of lipids. The Mexican axolotl (Ambystoma mexicanum) is a prominent source of the enzyme with a regeneration capacity in limbs. It has been shown that transfected human osteosarcoma and keratinocyte cells with epidermal lipoxygenase (LOXe) have an increased rate of cell migration. In the present study, LOXe, a peripheral membrane protein, was produced in Escherichia coli. The enzyme was purified using different detergents, anionic solutions, and gel filtration chromatography. Kinetic assay of the enzyme activity was carried out by the spectroscopy method using arachidonic acid as a substrate. Finally, the enzyme was characterized and its growth effect on human fibroblast cells was examined by MTT viability assay. Enzyme kinetic parameters including Km of 90.4 µM and Vmax of 2.63 IU were determined for LOXe. The enzyme with 0.1 nM end concentration promoted the growth of 5000 cells/well human fibroblast cells up to 11% (P < 0.01). In the present study, we introduce an E. coli expression system to produce an excessive amount of soluble LOXe and the efficient purification method to provide a soluble and active form of LOXe that is effective in stimulating human fibroblast cell proliferation.
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The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Abbreviations
- HETE:
-
Hydroxyeicosatetraenoic acid
- HFFF-PI6:
-
Human fibroblast cell line
- IU:
-
International unit
- IPTG:
-
Isopropyl β-d-1-thiogalactopyranoside
- LOX:
-
Lipoxygenase
- NP-40:
-
4-Nonylphenyl-polyethylene glycol
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
References
Baysal T, Demirdöven A (2007) Enzyme Microbial Technol 40(4):491–496
Klein RF, Allard J, Avnur Z, Nikolcheva T, Rotstein D, Carlos AS et al (2004) Science 303(5655):229–232
Manigrasso MB, O'Connor JP (2010) Actaorthopaedica 81(6):748–755
Song Y-S, Lee DH, Yu J-H, Oh D-K, Hong JT, Yoon D-Y (2016) Prostaglandins Other Lipid Mediators 123:1–8
Menzel-Severing J, Zenkel M, Polisetti N et al (2018) Sci Rep 8(1):10268
Stamm A, Strauß S, Vogt P, Scheper T, Pepelanova I (2018) Microbial Cell Fact 17(1):57
Fülbier A, Schnabel R, Michael S, Vogt PM, Strauß S, Reimers K et al (2014) Stem Cell Res Therapy 5(5):113
Menger B, Vogt PM, Allmeling C, Radtke C, Kuhbier JW, Reimers K (2011) Ann Surg 253(2):410–418
Tahan M, Movahedian-Attar A, Abbasian M, Mofid MR (2014) J Isfahan Med School 31(267):2162–2170
Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH et al (2009) Nature 460(7251):60–65
Haddad L, Babaeipour V, Mofid MR (2015) Res Pharm Sci 10(6):553–561
Gheysarzadeh A, Ansari A, Emami MH, Razavi AE, Mofid MR (2019) Pancreatology 19(3):429–435
Mittal M, Kumar RB, Balagunaseelan N, Hamberg M, Jegerschöld C, Rådmark O et al (2016) Bioorganic Med Chem Lett 26(15):3547–3551
Babaeipour V, Khanchezar S, Mofid MR, PesaranHagi AM (2015) Iran Biomed J 19(2):102–110
Krieg P, Fürstenberger G (2014) Biochem Biophys Acta 1841(3):390–400
Van Leyen K, Duvoisin RM, Engelhardt H, Wiedmann M (1998) Nature 395(6700):392–395
Brockes JP (1997) Science 276(5309):81–87
Lakshmi MC, Madhusudhan MC, Raghavarao KSMS (2012) Food Bioprocess Technol 5:193–199
Theerakulkait C, Barrett DM (1995) J Food Sci 60(5):1029–1033
Patel DD, Patel RR, Thakkar VR (2015) Appl Biochem Biotechnol 175(1):513–525
Soberman RJ, Harper T, Betteridge D, Lewis R, Austen K (1985) J Biol Chem 260(7):4508–4515
Flatman S, Hurst JS, McDonald-Gibson RG, Jonas GE, Slater TF (1986) Biochim Biophys Acta 883(1):7–14
Acknowledgements
The study described here was supported by Grant Number 394779 from the Isfahan University of Medical Science. The authors wish to appreciate Dr. Ali Gheysarzadeh for developmental discussions.
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Maryam Mashkouli: designed and performed the experiments, analyzed data and wrote the manuscript. Mahmoud Aghaei: designed, performed experiments and analyzed data. Mohammad Reza Mofid: designed and performed the experiments, analyzed data and wrote the manuscript.
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Mashkouli, M., Aghaei, M. & Mofid, M.R. Purification of Soluble Membrane-Bound Ambystoma mexicanum Epidermal Lipoxygenase from E. coli and Its Growth Effect on Human Fetal Foreskin Fibroblast. Protein J 39, 377–382 (2020). https://doi.org/10.1007/s10930-020-09898-w
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DOI: https://doi.org/10.1007/s10930-020-09898-w