RESUMO
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.
Assuntos
Aspergillus oryzae , Proteína Antagonista do Receptor de Interleucina 1 , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/química , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Interleucina-1/metabolismo , Receptores de Interleucina-1/metabolismoRESUMO
The mechanisms that pattern and maintain dendritic arbors are key to understanding the principles that govern nervous system assembly. The activity of presynaptic axons has long been known to shape dendrites, but activity-independent functions of axons in this process have remained elusive. Here, we show that in Caenorhabditis elegans, the axons of the ALA neuron control guidance and extension of the 1° dendrites of PVD somatosensory neurons independently of ALA activity. PVD 1° dendrites mimic ALA axon guidance defects in loss-of-function mutants for the extracellular matrix molecule MIG-6/Papilin or the UNC-6/Netrin pathway, suggesting that axon-dendrite adhesion is important for dendrite formation. We found that the SAX-7/L1CAM cell adhesion molecule engages in distinct molecular mechanisms to mediate extensions of PVD 1° dendrites and maintain the ALA-PVD axon-dendritic fascicle, respectively. Thus, axons can serve as critical scaffolds to pattern and maintain dendrites through contact-dependent but activity-independent mechanisms.
