New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5.
CCL5
CCR5
Chemokine activity testing
Protein purification
Recombinant chemokine production
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
15 10 2024
15 10 2024
Historique:
received:
12
07
2024
accepted:
04
10
2024
medline:
16
10
2024
pubmed:
16
10
2024
entrez:
15
10
2024
Statut:
epublish
Résumé
Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.
Identifiants
pubmed: 39406925
doi: 10.1038/s41598-024-75327-y
pii: 10.1038/s41598-024-75327-y
doi:
Substances chimiques
Chemokine CCL5
0
CCL5 protein, human
0
Receptors, CCR5
0
Recombinant Proteins
0
CCR5 protein, human
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
24188Subventions
Organisme : UKRI: Horizon Europe Guarantee Consolidator award
ID : EP/X023680/1
Organisme : UKRI: Horizon Europe Guarantee Consolidator award
ID : EP/X023680/1
Organisme : EPSRC
ID : EP/K039660/1
Informations de copyright
© 2024. The Author(s).
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