Cloning, expression and purification of recombinant dermatopontin in Escherichia coli.
Cell Movement
/ genetics
Chondroitin Sulfate Proteoglycans
/ biosynthesis
Cloning, Molecular
Endothelial Cells
/ metabolism
Escherichia coli
/ genetics
Extracellular Matrix Proteins
/ biosynthesis
Humans
Inclusion Bodies
/ genetics
Protein Folding
Recombinant Proteins
/ biosynthesis
Wound Healing
/ genetics
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2020
2020
Historique:
received:
16
08
2020
accepted:
09
11
2020
entrez:
30
11
2020
pubmed:
1
12
2020
medline:
8
1
2021
Statut:
epublish
Résumé
Dermatopontin (DPT) is an extracellular matrix (ECM) protein with diversified pharmaceutical applications. It plays important role in cell adhesion/migration, angiogenesis and ECM maintenance. The recombinant production of this protein will enable further exploration of its multifaceted functions. In this study, DPT protein has been expressed in Escherichia coli (E.coli) aiming at cost effective recombinant production. The E.coli GJ1158 expression system was transformed with constructed recombinant vector (pRSETA-DPT) and protein was expressed as inclusion bodies on induction with NaCl. The inclusion bodies were solubilised in urea and renaturation of protein was done by on-column refolding procedure in Nickel activated Sepharose column. The refolded Histidine-tagged DPT protein was purified and eluted from column using imidazole and its purity was confirmed by analytical techniques. The biological activity of the protein was confirmed by collagen fibril assay, wound healing assay and Chorioallantoic Membrane (CAM) angiogenesis assay on comparison with standard DPT. The purified DPT was found to enhance the collagen fibrillogenesis process and improved the migration of human endothelial cells. About 73% enhanced wound closure was observed in purified DPT treated endothelial cells as compared to control. The purified DPT also could induce neovascularisation in the CAM model. At this stage, scaling up the production process for DPT with appropriate purity and reproducibility will have a promising commercial edge.
Identifiants
pubmed: 33253286
doi: 10.1371/journal.pone.0242798
pii: PONE-D-20-25660
pmc: PMC7703894
doi:
Substances chimiques
Chondroitin Sulfate Proteoglycans
0
DPT protein, human
0
Extracellular Matrix Proteins
0
Recombinant Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0242798Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
Références
Biomed Res Int. 2013;2013:297305
pubmed: 23998121
Acta Crystallogr D Biol Crystallogr. 2007 Mar;63(Pt 3):295-301
pubmed: 17327666
J Investig Dermatol Symp Proc. 2000 Dec;5(1):40-6
pubmed: 11147674
Eur J Cell Biol. 2017 May;96(3):266-275
pubmed: 28336087
J Bacteriol. 1997 Jul;179(13):4403-6
pubmed: 9209061
Biochemistry. 2010 Jan 12;49(1):147-55
pubmed: 19928997
Anal Biochem. 1976 May 7;72:248-54
pubmed: 942051
Invest Ophthalmol Vis Sci. 2006 Aug;47(8):3303-10
pubmed: 16877395
Biochem J. 1999 Feb 1;337 ( Pt 3):537-41
pubmed: 9895299
Acta Biochim Biophys Sin (Shanghai). 2005 Apr;37(4):265-9
pubmed: 15806293
Appl Microbiol Biotechnol. 2006 Sep;72(2):211-22
pubmed: 16791589
J Biol Chem. 1989 Apr 5;264(10):5474-9
pubmed: 2925615
AMB Express. 2020 Mar 10;10(1):43
pubmed: 32152803
J Invest Dermatol. 2002 Sep;119(3):678-83
pubmed: 12230512
FEBS Lett. 1994 Sep 12;351(3):433-6
pubmed: 8082810
Appl Microbiol Biotechnol. 2010 Apr;86(4):1067-75
pubmed: 20012277
Protein Expr Purif. 2007 May;53(1):119-23
pubmed: 17194602
Adv Sci (Weinh). 2019 Mar 14;6(9):1801780
pubmed: 31065517
J Biol Chem. 1993 Sep 15;268(26):19826-32
pubmed: 8103522
J Biol Chem. 2011 Apr 29;286(17):14861-9
pubmed: 21398523
J Chromatogr A. 1997 Mar 7;764(1):27-33
pubmed: 9098994
Braz J Med Biol Res. 2004 Aug;37(8):1103-9
pubmed: 15273812
Protein Expr Purif. 2003 Feb;27(2):384-90
pubmed: 12597900
Sci Rep. 2014 Dec 09;4:7385
pubmed: 25486882
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Apr 15;818(1):29-33
pubmed: 15722041
Connect Tissue Res. 2006;47(4):177-89
pubmed: 16987749
Cells. 2019 Apr 09;8(4):
pubmed: 30970625
Matrix Biol. 2013 Jan;32(1):23-31
pubmed: 23262218
J Genet Eng Biotechnol. 2017 Jun;15(1):179-185
pubmed: 30647654
Protein Pept Lett. 2005 Jul;12(5):487-90
pubmed: 16029163