非表示:
キーワード:
Drosophila S2 cells; P13K; Protein purification; Single chain
要旨:
The variable fragment (Fv) of the monoclonal B1-8 antibody recognizes 3-nitro-4-hydroxy-phenylacetate (NP) and 5-iodo-NP (NIP) allowing for the affinity purification of the respective B cell antigen receptor with NP-sepharose and its specific elution with NIP-capronic acid (NIPcap). We generated an intracellular single-chain B1-8 Fv (iscFv), fused it to the N-terminus of the regulatory subunit (p85α) of phosphatidylinositol-3-kinase (PI3K) (isc-p85α), and examined the potential of this iscFv to serve as an intracellular elutable protein purification tag. The isc-p85α fusion protein could be specifically affinity-purified from the lysates of transfected Drosophila S2 cells with NP-sepharose and eluted with NIPcap, indicating the functional folding of the iscFv in the reducing environment of the cytosol. Furthermore, co-purification of the catalytic subunit of PI3K (p110) was achieved from lysates of co-transfected S2 cells as well as RBL-2H3 mast cells stably expressing isc-p85α. This indicates that the iscFv part of isc-p85α does not negatively influence p85α folding and interaction with p110. Moreover, successful incorporation of the p85α-moiety of isc-p85α into endogenous protein complexes in mast cells suggests the use of isc-containing fusion proteins for the native purification, elution, and analysis of intracellular signaling complexes.
