| PPD101 | |
| ID |  PPD101 |
| Project ID | PPD1 [Protein] [PREIMS] |
| Subject | Development of novel affinity tag system for the high-quality production of extracellular and membrane proteins |
| Project Theme | Development of novel affinity tag system for the high-quality production of extracellular and membrane proteins |
| Principal Investigator | Junichi Takagi |
| Affiliation | Institute for Protein Research, Osaka University |
| Backgrounds | - Mammalian cell expression is particularly suitable for the production of targeted proteins of mammalian origin - One problem with the recombinant production in stable mammalian cell lines is the lack of the established method to select and isolate a cell clone that highly expresses the targeted protein Protein purification is important for both functional characterization and crystallization |
| Highlights | - We developed a 21-residue peptide tag called TARGET tag and demonstrated its use for rapid stable cell line development and purification in recombinant production of a number of targeted proteins including NPP2, Sema6A and PlexinA2 - eTEV tag removable by the specific cleavage was developed as the second tag The tandem combination of the TARGET tag and the eTEV tag makes feasible the detection, quantification, purification and tag removal for a variety of proteins |
| Outline | Mammalian cell expression is particularly suitable for the production of targeted proteins of mammalian origin. One problem with the recombinant production in stable mammalian cell lines is the lack of the established method to select and isolate a cell clone that highly expresses the targeted protein. We have developed a peptide-based affinity tagging system that utilizes a P4 tag monoclonal antibody P20.1. The system is highly compatible with protein purification from cell culture supernatants. We applied this technology to develop a reliable method to select and isolate a cell clone that highly expresses the targeted protein. Both the sequence and the length of tag peptide were optimized, yielding a 21-residue peptide tag designated as TARGET tag. We have also developed a purification system utilizing eTEV tag as the second tag. Since eTEV tag is removable by the specific cleavage, the double tag system composed of the tandem combination of the TARGET tag and the eTEV tag makes feasible the detection, quantification, purification and tag removal for a variety of proteins. |
| Application | MPB3 MPB4 |
| Article | Protein Sci. (2008) Hybridoma. (2008) J Proteomics. (2010) |
| CSML File | PPD101.csml |