Abstract
This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays.
Keywords: Secreted protein, Purification, Mammalian, Ligand, Receptor
Background
E. coli is one of the organisms of choice for production of recombinant proteins from both prokaryotes and eukaryotes. The major advantages of bacterial expression systems are high productivity and low cost. However, mature proteins are essential for functional analyses (e.g., ligand binding assays). Most secreted eukaryotic proteins undergo post-translational modification by covalent glycan linkage and formation of disulfide bonds in the endoplasmic reticulum. These covalent modifications are essential for the general stability and folding of the secreted mature proteins. Therefore, the best method for production of mammalian secreted proteins is the use of a mammalian host to ensure the production of recombinant proteins that have undergone proper post-translational modifications. In the following protocol, we have described the detailed procedure for purification of secreted proteins from mammalian cells. This protocol is for production of FLAG-tagged proteins, but should be applicable to that of other tagged (e.g., His-tag) proteins as well.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from Chen et al. (2017). This work was supported by JSPS KAKENHI (Young Scientists A) Grant Number 16H06167, MEXT KAKENHI (Scientific Research on Innovative Areas) Grant Number 16H01194 (to E. I.); a European Molecular Biology Organization Fellowship (to C.C.); the Medical Research Council, UK; and the European Research Council (Advanced Grant 269058) grant to M.d.B.
References
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