Expression and production of pigment epithelium-derived factor (PEDF) and PEDF receptor variants from mammalian and bacterial cells.
Bacterial overexpression
Mammalian overexpression
PEDF
PNPLA2
Purification
Serpin
Journal
Protein expression and purification
ISSN: 1096-0279
Titre abrégé: Protein Expr Purif
Pays: United States
ID NLM: 9101496
Informations de publication
Date de publication:
06 2022
06 2022
Historique:
received:
16
10
2021
revised:
11
02
2022
accepted:
11
02
2022
pubmed:
20
2
2022
medline:
15
4
2022
entrez:
19
2
2022
Statut:
ppublish
Résumé
Human SERPINF1 gene codes for pigment epithelium-derived factor (PEDF), a secreted glycoprotein and member of the SERPIN superfamily. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. The cells produced and secreted recombinant PEDF proteins into the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. PEDF has affinity for PEDF-receptor (PEDF-R), a membrane-linked lipase encoded by the PNPLA2 gene. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3'end deletions and by induction with isopropyl β-d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. C-terminally truncated PEDF-R versions containing the ligand binding region retained the ability to bind PEDF. The data demonstrate that mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for further use in structural and biological studies.
Identifiants
pubmed: 35181508
pii: S1046-5928(22)00029-8
doi: 10.1016/j.pep.2022.106072
pmc: PMC8934279
mid: NIHMS1783345
pii:
doi:
Substances chimiques
Eye Proteins
0
Nerve Growth Factors
0
Receptors, Neuropeptide
0
Recombinant Proteins
0
Serpins
0
pigment epithelium-derived factor
0
pigment epithelium-derived factor receptor
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
106072Subventions
Organisme : Intramural NIH HHS
ID : Z01 DK015500
Pays : United States
Organisme : Intramural NIH HHS
ID : Z01 EY000306
Pays : United States
Organisme : Intramural NIH HHS
ID : ZIC DK015500
Pays : United States
Organisme : Intramural NIH HHS
ID : ZIB DK015500
Pays : United States
Organisme : Intramural NIH HHS
ID : ZIA EY000306
Pays : United States
Informations de copyright
Published by Elsevier Inc.
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