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Rho1D4 System for purifying membrane proteins
Nordic BioSite is exclusive distributor in Nordic Countries for PureCube Rho1D4 peptide, Agarose affinity resin and related products that make up a complete system for purifying proteins tagged with the epitope sequence of the rho1D4 antibody.
The PureCube system has been developed by Germany’s Cube Biotech as the first commercially available immunoaffinity resin for purification that uses Rho1D4 peptide in the elution buffer for competitive binding to the affinity resin, releasing tagged protein.
The Rho1D4 System to purify membrane proteins offers an ideal solution for membrane protein research, with its competitive binding providing gentler elution conditions than previous approaches such as changing pH.
The system also enables highly specific purification, along with higher yields, with binding capacity of up to 3-4 mg protein per mL of resin.
A principal 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 two acids prevents binding. In consequence, unspecific binding of proteins containing sequences similar to the rho1D4 epitope is minimized and the purity of the recovered protein is high.
A second 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 (See table below).
|Protein||Expression System||Purity||Yield||Purification steps|
|hCB2 G protein-coupled receptor||E.coli||90%||4.5 mg/5 L culture||Rho1D4 IAC + IMAC|
|hVN1R1G protein-coupled receptor||Inducible HEK293S cell line (mammal)||90%||1 mg/1 g cells||Rho1D4 IAC + SEC|
|FPR3 G protein-coupled receptor||Inducible HEK293S cell line (mammal)||90%||2 mg/6 g cells||Rho1D4 IAC + SEC|
|TAAR5 G protein-coupled receptor||Inducible HEK293S cell line (mammal)||90%||1 mg/9 g cells||Rho1D4 IAC + SEC|
|OR131-2 G protein-coupled receptor||Inducible HEK293S cell line (mammal)||90%||2.9 mg/10 g cells||Rho1D4 IAC + centrifugation|
|hOR17-4 G protein-coupled receptor||Inducible HEK293S cell line (mammal)||90%||7.5 mg/2.5 L culture||Rho1D4 IAC + SEC|
|hOR17-4 G protein-coupled receptor||Cell-free wheat germ extract||70%*||0.3 mg/mL reaction solution*||Rho1D4 IAC + SEC|
|CD81 Tetraspanin membrane protein||Inducible HEK293S cell line (mammal)||>95%||26 ug/3X10^7 cells||Rho1D4 IAC|
|AE1 Solute carrier||S. cerevisiae strain BJ5457||93%||2.5 mg/18 L culture||Rho1D4 IAC|
* Purity was achieved in one-step Rho1D4 IAC from low levels in the reaction solutions; yield was measured after eluate fractions were concentrated and applied to SEC column.
Further, the availability of complementary MagBeads, resin, antibody and pre-packed cartridges forms the basis for a complete rho1D4 toolkit that can be used in a wide range of functional studies, such as characterization of ligand binding and protein-protein interaction. Examples include:
- 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.
- 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.
- 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.
- 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.
Background: The Rho1D4 System
Rho1D4 refers to the last nine amino acids of the intracellular C-terminus of bovine rhodopsin. The name comes from the monoclonal antibody that specifically binds to the sequence. Combined with the rho1D4 antibody, this epitope can serve as a highly specific purification tag suitable for membrane proteins.
The illustration shows hypothetical fusion membrane protein with three transmembrane domains to show how a membrane protein of interest can be genetically modified to incorporate the rho1D4 tag with the sequence T-E-T-S-Q-V-A-P-A added to C-terminus. 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.
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. 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.
The PureCube system enables elution using the Rho1D4 peptide, pH or protease cleavage. The system offers an easy bind, wash and elute procedure involving just three steps:
Step 1: Bind
The rho1D4-tagged protein is incubated on a resin loaded with rho1D4 antibody, to sequester the target protein from the lysate.
Step 2: Wash
Washing away unwanted proteins and other lysate components leaves the target protein bound to the affinity matrix.
Step 3: Elution
Excess Rho1D4 peptide competitively binds to the matrix, with released target protein collected in the eluate.
Rho1D4 product categories
Nordic BioSite can provide conveniently aliquoted and priced PureCube Rho1D4 peptide, either for separate purchase or together with PureCube Rho1D4 Agarose to form Starter Sets. For detection in Western Blots, a highly specific Rho1D4 antibody is available, along with PureCube Rho1D4 MagBeads, recommended for dilute samples or pull-down experiments.
|143-16201||Rho1D4 peptide 5 mg|
|143-16203||Rho1D4 peptide 25 mg|
|143-33101||PureCube Rho1D4 Agarose 1 ml|
|143-33102||PureCube Rho1D4 Agarose 5 ml|
|143-33103||PureCube Rho1D4 Agarose 10 ml|
|143-33104||PureCube Rho1D4 Agarose 25 ml|
|143-33199||Starter Set 1: Rho1D4 Agarose + Rho1D4 peptide 5 mg|
|143-33201||PureCube Rho1D4 MagBeads 1 ml|
|143-33205||PureCube Rho1D4 MagBeads 5 ml|
|143-33225||PureCube Rho1D4 MagBeads 25 ml|
|143-33299||Starter Set 2: Rho1D4 MagBeads + Rho1D4 peptide 5 mg|
|143-33301||PureCube Rho1D4 Cartridge 1×1 ml|
|143-33305||PureCube Rho1D4 Cartridge 1×5 ml|
|143-33399||Starter Set 3: Rho1D4 Cartridge + Rho1D4 peptide 5 mg|
|143-40020||Rho1D4 Antibody 200 ug|