What is rho1D4

What is rho1D4?

Rho1D4 refers to the last 9 amino acids of the intracellular C-terminus of bovine rhodopsin. The name comes from the monoclonal antibody that specifically binds to the sequence.(1) Combined with the rho1D4 antibody, this epitope can serve as a highly specific purification tag suitable for membrane proteins. A membrane protein of interest can be genetically modified to incorporate the rho1D4 tag at the C-terminus (Fig. 1). Once outfitted with this sequence, the target protein can be captured on an affinity matrix loaded with rho1D4 antibody and subsequently eluted by adding an excess of rho1D4 peptide to competitively bind with the matrix antibody. This provides for gentler elution conditions than, for example, changing pH.
Fig. 1: A hypothetical membrane protein with 3 transmembrane domains. The rho-1D4 tag, with the sequence T-E-T-S-Q-V-A-P-A, has been added to the C-terminus.
Step 1: Bind. Incubating the rho1D4-tagged protein on a resin loaded with rho1D4 antibody sequesters the target protein from the lysate.
 

The rho1D4 System

The rho1D4 epitope and antibody pair was first characterized in the 1980's and used to purify bovine rhodopsin expressed in monkey kidney cells by coupling the antibody to Sepharose® beads.(1,2) Since then, the rho1D4 system (tag, antibody-coupled affinity matrix, eluent peptide) has been used to study a handful of membrane proteins including G-protein coupled receptors (GPCR), an ATP-binding cassette transporter, a solute counter-transporter, and a tetraspanin membrane protein. One advantage of the system is the high specificity of the antibody-epitope interaction. Epitope sequence and chain length are critical for binding. For example, replacing the third alanine with glycine which removes a single methyl group, eliminates binding. Likewise, the full 9-amino acid tag binds tightest to the rho1D4 antibody and removing 2 amino acids prevents binding.(1) As a consequence, unspecific binding of proteins containing sequences similar to the rho1D4 epitope is minimized and the purity of the recovered protein is high (Table 1). (3,4,5,6,7,8,14)
Step 2: Wash. Unwanted proteins and other lysate components are washed away, leaving the target protein bound to the affinity matrix.
 

Advantages of the rho1D4 system

Therefore the sequence of the 1d4 tag is highly specific and only present in rhodopsin and related photoreceptor proteins which minimizes unspecific binding. In addition it is not necessary to insert multiple copies of the 1d4 tag like it is required for Flag tags (15). Another advantage is the high yield of the eluted target protein. Expression systems, including bacterial, yeast, and mammalian cell lines, have been optimized for a selection of GPCRs and other membrane proteins. Purification of the membrane proteins was done with the rho1D4 system followed by gel filtration or centrifugal concentration to remove the eluent peptide. In all systems authors reported recovery of milligram amounts of protein (Table 1).(3,4,5,6,7,8) Finally, the purified proteins can be used for functional studies, such as characterization of ligand binding and protein-protein interaction. For example, tagged ABCA4 was immobilized to a matrix loaded with rho1D4 antibody to characterize its binding affinity for its natural ligand, an adduct of retinal, and subsequent release by addition of ATP.(2) In another example, the protein CD81 was immobilized in its entirety to plates coated with rho1D4 antibody and shown to exhibit the same binding affinity for Hepatitis C Virus envelope E2 protein as the isolated soluble fragment of the protein.(3). . Ligand binding to the Cannabinoid receptor 2 (CB2) was analyzed by SPR and radiolabel exchange. It was also possible to perform G protein activation assays with CB2 reconstituted into liposomes (14). Lastly, vesicles reconstituted with the membrane domain of the anion exchange AE1 tagged and purified with rho1D4 system showed the same rate of sulfate efflux as vesicles with erythrocyte-sourced AE1.(6)
Step 3: Elution. Excess rho1D4 peptide competitively binds to the matrix and the released target protein is collected in the eluate.
 

Usage of the rho1D4 system in the literature

ProteinExpression SystemPurityYield (total protein recovered)Purification steps taken
hCB2 G protein-coupled receptor14 E.coli 90% 4.5 mg/5 L culture Rho1D4 IAC + IMAC
hVN1R1 G protein-coupled receptor9 Inducible HEK293S cell line (mammal) 90% 1 mg/1 g cells Rho1D4 IAC + SEC
FPR3 G protein-coupled receptor8 Inducible HEK293S cell line (mammal) 90% 2 mg/6 g cells Rho1D4 IAC + SEC
TAAR5 G protein-coupled receptor7 Inducible HEK293S cell line (mammal) 90% 1 mg/9 g cells Rho1D4 IAC + SEC
OR131-2 G protein-coupled receptor5 Inducible HEK293S cell line (mammal) 90% 2.9 mg/10 g cells Rho1D4 IAC + centrifugation
hOR17-4 G protein-coupled receptor11 Inducible HEK293S cell line (mammal) 90% 7.5 mg/2.5 L culture Rho1D4 IAC + SEC
hOR17-4 G protein-coupled receptor12 Cell-free wheat germ extract 70%* 0.3 mg/mL reaction solution* Rho1D4 IAC + SEC
CD81 Tetraspanin membrane protein3 Inducible HEK293S cell line (mammal) >95% 26 ug/3X10^7 cells Rho1D4 IAC
AE1 Solute carrier6 S. cerevisiae strain BJ5457 93% 2.5 mg/18 L culture Rho1D4 IAC
Table 1. Examples of purity and yield reported in the literature for membrane proteins purified with the rho1D4 system. Although many of the proteins purified with the rho1D4 system have been G protein-coupled receptors, the system has the flexibility to facilitate characterization of other membrane proteins such as transporters. Referenced articles are listed in the literature cited. IAC: immunoaffinity chromatography; IMAC: immobilized metal affinity chromatography; SEC: size-exclusion chromatography.

Getting started with the rho1D4 System

Using the rho1D4 system to purify membrane proteins requires some protocol optimization, as each protein behaves differently once expressed in a heterologous host, in response to solubilization detergents, and within the purification column. Important is to have all the items necessary and to screen for optimal protocol conditions and steps. Below are the 4 items needed and each is accompanied by protocols or information to help you implement the rho1D4 system quickly and efficiently. If we can help with the optimization, please contact us.
 
 
Your target protein needs to be fused to the rho1D4-tag. You can do this on the C- or on the N-terminus, depending on where the tag interferes the least with protein activity. These protocols outline primer design (with copy-paste sequences) for the necessary expression constructs.
Cloning Strategy and Primer Design for C-­terminal Rho­1D4 Fusion Proteins
Cloning Strategy and Primer Design for N-terminal Rho1D4 Fusion Proteins
 
Prokaryotic and eukaryotic systems offer different advantages when expressing your target protein. Selection of an appropriate expression system requires some trial-and-error and screening for optimal expression conditions. This protocol details the optimization of host, media, temperature, and induction time for protein expression in bacterial systems (E. coli).
Screening for Optimal Expression Conditions using E. coli Expression Systems
 
Detergents are used to solubilize membrane proteins and selecting the optimal detergent for your target protein is critical for subsequent purification. This protocol outlines the screening of 7 commonly used detergents.
Screening Detergents for Optimal Solubilization and Purification of Membrane Proteins
 
An affinity matrix coupled to the rho1D4 antibody and the rho1D4 peptide to serve as a competitive eluent are needed to purify your rho1D4-tagged protein. PureCube Rho1D4 Agarose and Rho1D4 peptide can be purchased together as a starter set or individually to meet your throughput needs. For detection in Western Blots, a highly specific Rho1D4 antibody is available. This protocol outlines the solubilization and purification process.
Purification of rho1D4-tagged Membrane Proteins Using PureCube Rho1D4 Agarose
Purification of rho1D4-tagged Membrane Proteins Using PureCube Rho1D4 MagBeads

1. Hodges, R.S., et al. 1988. Antigen-antibody interaction. J Biol Chem 263: 11768-11775.
2. Oprian, D.D., et al. 1987. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc Natl Acad Sci USA 84: 8874-8878.
3. Takayama, H. et al. 2008. High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81. PLoS ONE 3: e2314 (DOI: 10.1371/journal.pone.0002314).
4. Zhong, M. and Molday, R.S. 2010. Biding of retinoids to ABCA4, the photoreceptor ABC transporter associated with Stargardt Macular Degeneration. Methods Mol Biol 652: 163-176.
5. Leck, K.-J., et al. 2010. Study of bioengineered zebra fish olfactory receptor 131-2: receptor purification and secondary structure analysis. PLoS ONE 5: e15027(DOI:10.1371/journal.pone.0015027).
6. Bonar, P. and Casey, J.R. 2010. Purification of functional human Cl-/HCO3- exchanger, AE1, over-expressed in Saccharomyces cerevisiae. Protein Express Purif 74: 106-115.
7. Wang, X. et al. 2011. Study of two G-protein coupled receptor variants of human trace amine-associated receptor 5. Sci Rep1: 102 (DOI:10.1038/srep00102).
8. Wang, X. and Zhang, S. 2011. Production of a bioengineered G-protein coupled receptor of human formyl peptide receptor 3. PLoS ONE 6: e23076 (DOI: 10.1371/journal.pone.0023076).
9. Corin, K. et al. 2011. Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1. Sci Rep 1: 172 (DOI: 10.1038/srep00172).
10. Kaiser, L., et al. 2008. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand biding analysis. Proc Natl Acad Sci USA 105: 15726-15731.
11. Cook, B. L., et al. 2008. Study of a synthetic human olfactory receptor 17-4: expression and purification from an inducible mammalian cell line. PLoS ONE 3: e2920 (DOI: 10.1271/journal.pone.0002920).
12. Cook, B. L., et al. 2009. Large-scale production and study of a synthetic G protein-coupled receptor: human olfactory receptor 17-4. Proc Natl Acad Sci USA 106: 11925-11930.
13. Zhong, M., et al. 2009. Role of the C terminus of the photoreceptor ABCA4 transporter in protein folding, function, and retinal degenerative diseases. J Biol Chem 284: 3640-3649.
14. Locatelli-Hoops, S.C. et.al. 2013. Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2. Biochim. Biophys. Acta 1834 (10):2045-56.
15. Molday, L. L., & Molday, R. S. (2014). 1D4: a versatile epitope tag for the purification and characterization of expressed membrane and soluble proteins. In (pp. 1-15). Humana Press, New York, NY.
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