A Carboxy-Terminal Affinity Tag for the Purification and Mass Spectrometric Characterization of Integral Membrane Proteins

Abstract

G-protein-coupled receptors (GPCRs) and other, structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here the implementation of an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tagspecific, high-affinity monoclonal antibody. Expressing two GPCRs as well as two related ATP binding cassette (ABC) transporters in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 in an annotated spectrum library, and to identify the N-glycosylation sites for CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.

Differential Phospholipid Substrates and Directional Transport by ATP-binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants

“…The immunoaffinity purification and reconstitution methodology used here to characterize ABCA1, ABCA4, and ABCA7 have broad application for analysis of other ABC transporters as well as other membrane proteins (58,59). It will be of particular interest to identify the putative lipid substrates and direction of transport for ABCA3 associated with neonatal surfactant deficiency and pediatric interstitial lung disease and ABCA12 linked to harlequin ichthyosis as well as other members of the ABCA subfamily, including ABCA2.…”

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…The 1D4 epitope is a short hydrophilic sequence (TETSQVAPA) derived from the C-terminus of bovine rhodopsin [99,100]. Combining this epitope and the high affinity rho-1D4 monoclonal antibody (available from www.flintbox.com) has established useful tools in antibody-based purification, localization studies, and western blot analysis of 1D4-tagged membrane proteins [101,102].…”

The 1D4 Antibody Labels Outer Segments of Long Double Cone But Not Rod Photoreceptors in Zebrafish

“…10,11 Given its high specificity, it has been widely used in studies addressing structure and function of rhodopsin, 12 as well as a reliable fusion tag for purification of nonrelated receptors. 13,14 In mice, it has been used for the detection of rhodopsin 15 and its subcellular distribution 16 during retinal degeneration. This antibody is commercially available (ab5417; Abcam, Cambridge, UK; R5403; Sigma-Aldrich, St. Louis, MO; sc-57,432; SCBT, Santa Cruz, CA; MAB5356; Millipore, Billerica, MA) and according to the supplier is used to detect rhodopsin also in zebrafish.…”