Micropreparation of a Native PHGPx Protein From Radish Seedlings by Immunoaffinity Chromatography
- VernacularTitle:用免疫亲和层析法纯化萝卜PHGPx天然蛋白
- Author:
Xiaodong YANG
;
Jinyuan LIU
- Publication Type:Journal Article
- Keywords:
immunoaffinity chromatography;
native protein;
polyclonal antibodies;
phospholipid hydroperoxide glutathione peroxidase;
radish
- From:
Progress in Biochemistry and Biophysics
2005;32(8):794-799
- CountryChina
- Language:Chinese
-
Abstract:
Radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) was identified as a mitochondrion-targeting PHGPx in previous work. To determine its cleavage site of the targeting peptide, the immunoaffinity chromatography (IAC) purification approach was carried out to isolate the native RsPHGPx protein.Polyclonal antibodies directed against recombinant RsPHGPx were raised in rabbit. Monospecific anti-RsPHGPx antibodies were isolated by means of affinity chromatography using the recombinant RsPHGPx as affinity ligand, and employed in assembling an IAC column. A single-step, highly specific and easy-to-use protocol was developed for purification of the active RsPHGPx protein through the assembled IAC column. Using this approach, a specific protein of the expected molecular size was obtained from the mitochondrial fraction of radish seedlings. Western blot analysis showed that it could be specifically recognized by anti-RsPHGPx antibodies, and an enzyme activity assay indicated that it exhibited significant PHGPx activity, suggesting that the purified protein should be the desired native RsPHGPx. These results will lead to clarification of the targeting peptide and the active mature protein of RsPHGPx and will be helpful to further probe the intracellular localization mechanism and biological fun ction of this plant PHGPx.
