원문정보
초록
영어
The newly discovered Th2 pro-inflammatory cytokine, interleukin-17E belongs to the member of IL-17 family. In this study, bioactive recombinant mutated human IL–17E (rhIL–25) was synthesized using overlapping PCR strategy and amino acid mutations were carried out using site directed mutagenesis. Four cysteins at 78th, 83rd, 136th and 138th positions were involved in disulphide bond formation and were responsible for biological activity of mature protein. These four cysteins were replaced with serine using nucleotide substitution and the desired outcome was cloned into expression vector pRSET-A followed by expressed in a salt inducible Escherichia coli GJ1158. The transformants were selected by ampicillin resistance marker and also by DNA sequencing. SDS–PAGE analysis confirms 17.06 kDa purified protein against low molecular weight protein marker. Protein quantification was carried out using Lowry’s method. Approximately 104 mg/L of recombinant IL-17E was produced at 37 ℃. Biological activity of protein was determined by the release of IL–6 from PBMC cells using rhIL–17E. This is the first report on production of interleukin-17E structural variant with enhanced specific activity without compromising the biological activity.
목차
1. Introduction
2. Material and Methods
2.1. Microbial Strains and Plasmid
2.2. Medium Composition
2.3. Gene Amplification using PCR
2.4. Site Directed Mutagenesis
2.5. Cloning of Structural Variant into Expression Plasmid pRSET-A
2.6. Heterologous Expression using E.coli GJ1158
2.7. Purification of IL-17E structural variant
2.8. Biological Activity of rhIL–25
3. Results and Discussion
3.1. Amplification of Mutated rh17E
3.2. Generation of Recombinant DNA
3.3. Expression of rh17E structural variant
3.4. Purification of rhIL-17E structural variant
3.5. Biological Activity Assay
4. Conclusion
Acknowledgements
References