Preparation and functional analysis of truncated fve
Date Issued
2011
Date
2011
Author(s)
Chang, Yu-Min
Abstract
FIP-fve, a fungal immunomodulatory protein isolated from Flammulina Velutipes, has been reported to be a dimer in its native form. The main driving force of fve dimerization was the hydrophobic interactions between the N-terminal alpha-helices of the two monomers. The fve dimer was further stabilized by the beta-sheet which consisted of the N-terminal beta-strands of the two monomers. Previous study also suggested that fve was a superantigen-like immunomodulatory protein, which activated T cells through the binding of both MHC molecules and the T cell receptor. Our objective in this study was to prove that the dimerization was critical and necessary for the immunomodulatory activity of fve. This study included two parts: (1) to study the relationship between fve dimer and its immunomodulatory activity and (2) to investigate the expiration date of native fve. We also established a sandwich enzyme-linked immunosorbent assay (ELISA) to determine the concentration of fve.
To study the relationship between fve dimer and its immunomodulatory activity, we used E.coli expression system to produce seven truncated fve. However, only truncated fve-28-103, which could not form dimer due to the deletion of 1- 27 residues at the N-terminus, was successfully expressed and purified. The fve-28-103 was then labeled with FITC to confirm its binding activity with peritoneal macrophages and T cells. We also detected the IFN-gamma production by murine splenocytes which were induced by truncated fve-28-103. Our results showed that the binding ability of truncated fve-28-103 was lower than that of native fve toward peritoneal macrophages. The binding was not significant on T cells. Moreover, truncated fve-28-103 did not activate splenocytes to produce IFN-gamma. According to these results, we concluded that the dimerization was essential for the activity of fve.
We have evaluated the expiration date of native fve in food under different sterilization processes and storage conditions. The SDS-PAGE analysis showed that little degradation of fve was observed after the treatment with 121℃ for 20 minutes or 138℃ for 18 seconds. Then, the fve could be stored at 25℃ at least for 6 months. Fve treated with 121℃ for 20 minutes or 130℃ for 12 seconds could also be stored at 4℃ at least for 6 months. To develop an analytical method for fve, we have further established a specific and sensitive sandwich ELISA for fve quantification using anti-fve monoclonal antibodies. The linear quantitative concentration ranged from 103-106 pg/mL with a multiple coefficient of determination r2=0.9403. As a result, fve could be stored at least for 6 months under different treatments. This fve ELISA method could be applied for the determination of fve content in food samples.
Subjects
superantigen
GST gene fusion system
immunomodulation
enzyme-linked immunosorbent assay
Type
thesis
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