Patent Application
20080097084
The present invention overcomes the problems of purification of membrane proteins such as the family of G-protein coupled receptors (GPCRs) by providing an epitope tag derived from rhodopsin. Rhodopsin is a prototypical member of the GPCR family of transmembrane proteins. Thus, this invention takes advantage of the properties of the amino-terminal extracellular region of rhodopsin as both an epitope site (an identification peptide, or tag) and as a peptide that assists in inserting the protein into the cell membrane. The invention is directed towards enhanced expression, purification and detection of target polypeptides, in particular members of the GPCR family of proteins. Fusing the amino-terminus of a target protein to the amino terminal extracellular region of the GPCR protein rhodopsin (RHO) produces a hybrid polypeptide which can be expressed at high levels and which is bound by antibodies to rhodopsin.
The following examples are intended to convey certain principles of the invention. These examples are not intended to limit the scope of the claims to any particular example. It is understood that the claims are to be given their broadest reasonable interpretation in view of the description herein, any prior art, and the knowledge of those of ordinary skill in the field. Those skilled in the art will readily appreciate that many variations may be derived using the following description.
This invention responds to the need to express and purify proteins and polypeptides that span or are associated with the plasma membrane. Target polypeptides are proteins of interest to the user, and include primarily transmembrane proteins such as the GPCR family of proteins. However, this invention is not limited to GPCR proteins, or even to complete (full-length) proteins. For the purpose of this invention, target polypeptide refers to a protein or portion of a protein that spans or associates with the plasma membrane. Since the identification polypeptide (the epitope tag) is derived from rhodopsin, homology among the GPCR family of proteins suggests that this invention is ideal for expressing and purifying members of the GPCR family.
There are many forms of rhodopsin, both within and across species. One of the best-studied rhodopsin molecules is bovine rhodopsin. The first 36 amino acids of the bovine rhodopsin (RHO) protein are extracellular, and may assist in inserting the rhodopsin protein in the membrane. The first 20 amino acids from the amino-terminus of the bovine RHO protein (MNGTEGPNFY VPFSNKTGW) (SEQ ID NO: 1) can be readily incorporated at the amino-terminus of a target polypeptide of interest, and expressed in cell culture. This polypeptide sequence makes a good epitope tag because it is extracellular, and therefore accessible, and also because it is believed to include a cryptic site assisting in membrane insertion. Furthermore, because rhodopsin is a prototypical member of the GPCR family, the RHO tag is unlikely to interfere with processing or folding of other transmembrane proteins. Thus, the amino-terminal portion of bovine rhodopsin also aids in expressing functional tagged protein on the cell membrane.
There are also many antibodies that can specifically recognize the amino-terminal domain of bovine rhodopsin. See D. Hicks and R. S. Molday, Exp. Eye Res. 1986, 42 (1):55-71, P. Rohlich et al, Exp. Eye Res. 1989, 49 (6):999-1013, P. A. Hargrave et al, Exp. Eye Res. 1986, G. Adanus et al, Vision Res. 1991, 31 (1):17-31 and G. Adamus et al, Pept. Res. 1988, 1 (1):4247. Antibodies, particularly monoclonal antibodies, directed against the first 15 amino-terminal peptides of bovine rhodopsin (MNGTEGPNFY VPFSN) (SEQ ID NO: 2) are highly effective for identifying hybrid polypeptides tagged with the first 20 amino acids from the amino-terminus of bovine rhodopsin. However, antibodies directed against the amino-terminal regions of other forms of rhodopsin would also apply to this invention. It is important that the antibody bind with high affinity to the rhodopsin identification peptide portion of the hybrid polypeptide. Usually this will mean that the antibody was directed against all or a portion of the identification peptide.
Alternative embodiments of the invention contemplate using the amino terminal extracellular domains of rhodopsin from any other species. Examples include rat rhodopsin, human rhodopsin, mouse rhodopsin or fruit fly rhodopsin. For the purposes of this invention, the amino terminal extracellular domain of rhodopsin refers to the most amino terminal portion of rhodopsin which is substantially extracellular when expressed endogenously. The amino terminal extracellular domain is not limited to the entire extracellular region of the amino terminal region, but encompasses a portion or subpart of the extracellular region of the amino terminal region.
Purification of the transmembrane proteins is conceptually straightforward. For example, if the target polypeptide is a member of the GPCR family, it is first fused to the rhodopsin identification peptide to create the hybrid polypeptide described above. The hybrid polypeptide is made from the peptide sequence of the GPCR target polypeptide by ligating the nucleotide sequence encoding the GPCR protein with the nucleotide sequence coding for the first 20 amino acids of bovine rhodopsin. The sequences should be joined such that the nucleotides coding the amino-terminus of rhodopsin will be linked to the amino-terminus of the GPCR target polypeptide. The resulting hybrid polynucleotide (coding for the hybrid polypeptide) is then inserted into an expression vector capable of expressing in cultured cells, such as Sigma's FLAG expression vector (Sigma-Aldrich Corp., St. Louis, Mo.). Cultured cells can then be transformed with the plasmid, and induced to express the hybrid polynucleotide.
The expressed protein can be purified using an antibody having a high specificity and affinity for the rhodopsin tag. This example uses a monoclonal antibody raised in mouse and directed against the first 15 amino acids of bovine rhodopsin (SEQ ID NO: 2). The antibody is conjugated to a matrix, such as a resin like sepharose, and a solution containing the hybrid polypeptide harvested from the cultured cells is applied to the matrix. The exposed amino-terminal portion of the hybrid polypeptide binds to the antibody, and is held in the matrix. The resin can then be washed to remove impurities. Finally, the hybrid polypeptide is eluted from the resin. Depending on the resin and desired stringency of elution, different ways of eluting the hybrid polypeptide may be used. For example, the hybrid polypeptide can be eluted by pH gradient, salt gradient or detergent. Alternatively, the hybrid polypeptide could be competed off of the antibody using a small peptide. For this example, an excess of peptide expressing the first 15 amino acids of rhodopsin could be used to elute the hybrid polypeptide described above.
Purified hybrid polypeptide can then be analyzed. This invention is particularly directed at aiding in the creation of protein crystals for protein structure determination by crystallographic techniques. Because the amino-terminal rhodopsin tag is small, it should not alter or complicate the crystal structure determination. Bovine rhodopsin also has the advantage that it has been crystallized, and therefore the structure of the amino-terminal domain is known.
It is also possible to detect the hybrid polypeptide after it has been expressed. For example, the rhodopsin marker can be used to monitor cell-surface expression of hybrid polypeptides such as membrane proteins. A hybrid protein made as described above by fusing a rhodopsin marker (identification peptide) to a GPCR protein can be visualized using an antibody to the RHO tag. Cells transformed by the plasmid containing the hybrid polypeptide can be immunolabled to verify that the proteins are being expressed in the plasma membrane in adequate levels. Since the 20 amino acid tag is expressed extracellularly, it is also potentially useful as a vital marker in living cells, as well as fixed. Further, surface and total expression can be quantified by binding assays with radiolabeled antibodies. Visual immunohistochemistry using the anti-rhodopsin antibody to recognize the rhodopsin tag on the hybrid polypeptide is can also be used to quantify surface or internal expression. Hybridization blotting using an antibody to the rhodopsin tag (e.g. Western blotting) is another way to determine expression of the hybrid polypeptide.
Virtually any reporting agent can be used in conjunction with the antibody against the rhodopsin tag. Enzymatic reporting agents such as horseradish peroxidase (HRP ) would be effective. As described above, radioactive labels, and fluorescent labels are also contemplated. The reporting agent could be either conjugated or incorporated directly into the anti-rhodopsin antibody. Alternatively, the reporting agent could be linked to the antibody/hybrid protein complex through a secondary antibody. If the antibody against the rhodopsin tag was raised in mouse, a secondary antibody against mouse IgG labeled with a reporting agent could be used to visualize the hybrid polypeptide.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US04/31831 | 9/27/2004 | WO | 00 | 11/26/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60506304 | Sep 2003 | US |
