Ligand and Protein Fishing with Heat Shock Protein 90 Coated Magnetic Beads

Abstract

Heat shock protein 90α (Hsp90α) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90α has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90α ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90α coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins.Heat shock protein 90 (Hsp90) is a family of cellular proteins (Hsp90α, Hsp90β, Grp94, Trap1) that act as molecular chaperones, which guide the normal folding, intracellular disposition, and proteolytic turnover of many key regulators of cell growth and survival. 1 It is one of the most abundant molecular chaperones in eukaryotes 2 and is essential in the activation of over 100 client proteins, including receptors, protein kinases, and transcription factors. 1,3 Increased expression and activity of Hsp90 have been observed in human cancers, and this protein has become a therapeutic target for the development of new anticancer agents. 4 The intrinsic ATPase activity of Hsp90 is also a target in drug discovery as ATPase activity is a key component in the chaperone function. 5Although a large number of Hsp90 client proteins have been identified, it is likely that there are many as of yet unidentified client proteins whose function could help elucidate novel oncogenic signaling pathways. At the present time, co-immunoprecipitation (IP) is the primary method used to identify client proteins. 6 However, in this approach an antibody raised against the coprecipitating protein is required for identification by Western blot analysis. Thus, it is difficult to find new proteins without first knowing what you are looking for. Additional problems with this technique include antibody contamination, nonspecific binding interactions, and leaching contamination that can result from protein A or protein G immobilization on agarose.We now report an alternative approach to the isolation of Hsp90 client proteins based upon the immobilization of Hsp90α on the surface of magnetic beads. In a previous study, we demonstrated that Hsp90 could be immobilized on a silica-based stationary phase through

Magnetic solids in analytical chemistry: A review

“…This method is based on the coordination of transition metal ions such as Cu 2+ , Ni 2+ , Zn 2+ and Co 2+ with imidazole, thiol, and indole groups present in the protein chains [19,[96][97][98]. Moreover, these methods provide a fast, simple, and sensitive way to detect protein based on competitive immunoassays using magnetic nanoparticles under magnetic fields [99][100][101][102][103][104][105][106]. Finally, it should be highlighted that the immobilization of metal ions on the magnetic silica particle surface improves the enrichment or pre-concentration process [107].…”

The preparation and development of cellular membrane affinity chromatography columns

“…In addition, we have shown previously that protein-coated magnetic beads can be successfully used to separate small-molecule binders from nonbinders 33,34 , as well as proteins from cellular extracts 34 . Preliminary studies with magnetic beads coated with cellular membranes from the KXα3β4R2 cell line expressing the α3β4 nAChR demonstrate that transmembrane proteins can also be studied in this manner (R.M., unpublished data).…”

Screening bioactive compounds from Ligusticum chuanxiong by high density immobilized human umbilical vein endothelial cells

“…The major issue of these researches is the relative lack of specificity. Receptors, the main target for drugs, are also immobilized on silica gel to construct more specific receptor-based stationary phase for screening bioactive compounds from traditional medicines [42][43][44]. Although the specificity improved greatly, the receptor-based stationary phase has limitations because it is difficult to maintain the natural conformation of the receptor without a bilayer membrane system.…”