Expression and characterization of recombinant IL-1Ra in Aspergillus oryzae as a system.
Anakinra
Aspergillus oryzae
Half-life
IL-1Ra
Interleukin-1 receptor antagonist
Surface plasmon resonance
Journal
BMC biotechnology
ISSN: 1472-6750
Titre abrégé: BMC Biotechnol
Pays: England
ID NLM: 101088663
Informations de publication
Date de publication:
20 06 2023
20 06 2023
Historique:
received:
20
02
2023
accepted:
08
06
2023
medline:
22
6
2023
pubmed:
21
6
2023
entrez:
20
6
2023
Statut:
epublish
Résumé
The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae. We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability than E. coli IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM). This study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli IL-1Ra.
Sections du résumé
BACKGROUND
The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae.
RESULTS
We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability than E. coli IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM).
CONCLUSION
This study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli IL-1Ra.
Identifiants
pubmed: 37340430
doi: 10.1186/s12896-023-00785-7
pii: 10.1186/s12896-023-00785-7
pmc: PMC10283290
doi:
Substances chimiques
Interleukin 1 Receptor Antagonist Protein
0
Recombinant Proteins
0
Interleukin-1
0
Receptors, Interleukin-1
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
15Informations de copyright
© 2023. The Author(s).
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