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Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli
- Source :
- Protein expression and purification. 89(2)
- Publication Year :
- 2013
-
Abstract
- The C5aR antagonist (C5aRA) 1 , which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli . To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37 °C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496 mg/L of NusA-fused C5aRA could be produced.
- Subjects :
- Recombinant Fusion Proteins
Genetic Vectors
Gene Expression
Biology
medicine.disease_cause
C5a receptor
Maltose-Binding Proteins
Bioreactors
Thioredoxins
medicine
Escherichia coli
Humans
Anaphylatoxin
Cloning, Molecular
Receptor
Overproduction
Receptor, Anaphylatoxin C5a
Escherichia coli Proteins
Biological activity
Peptide Elongation Factors
Fusion protein
Biochemistry
Solubility
Thioredoxin
Transcriptional Elongation Factors
Biotechnology
Plasmids
Transcription Factors
Subjects
Details
- ISSN :
- 10960279
- Volume :
- 89
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Protein expression and purification
- Accession number :
- edsair.doi.dedup.....815cef4eda4c4e9f9369358bcec84e85