Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-02-11
2004-12-14
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S412000, C530S350000, C530S300000, C435S069100, C435S007900, C435S007100, C549S207000, C568S411000, C562S830000
Reexamination Certificate
active
06831160
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to affinity purification of proteins and more specifically to the use of a modified bis-arsenical fluorescein compound immobilized to a solid support for protein purification.
BACKGROUND OF THE INVENTION
Many techniques in the biological sciences require attachment of labels to molecules, such as polypeptides. For example, the location of a polypeptide within a cell can be determined by attaching a fluorescent label to the polypeptide.
Traditionally, labeling has been accomplished by chemical modification of purified polypeptides. For example, the procedures for fluorescent labeling require that the polypeptide be covalently reacted in vitro with a fluorescent dye, then repurified to remove excess dye and/or any damaged polypeptide. Using this approach, problems of labeling stoichiometry and disruption of biological activity are often encountered. Furthermore, to study a chemically modified polypeptide within a cell, microinjection can be required. This can be tedious and cannot be performed on a large population of cells.
Thiol- and amine-reactive chemical labels exist and can be used to label polypeptides within a living cell. However, these chemical labels are promiscuous. Such labels cannot specifically react with a particular cysteine or lysine of a particular polypeptide within a living cell that has numerous other reactive thiol and amine groups.
A more recent method of intracellular labelling of polypeptides in living cells has involved genetically engineering fusion polypeptides that include green fluorescent protein (GFP) and a polypeptide of interest. However, GFP is limited in versatility because it cannot reversibly label the polypeptide. The ability to generate a wide range of specifically labeled molecules easily and reliably would be particularly useful.
The use of genetically-encoded affinity tags is now a standard method of purifying proteins (reviewed in (Hannig and Makrides, 1998; LaVallie and McCoy, 1995; Makrides, 1996; Nilsson, et al., 1997 ; Uhlén and Moks, 1990)). This technique allows for simple purification of a protein of interest by fusing to it a tag with affinity for a stationary phase. Most affinity tags are small-molecule binding proteins (e.g. maltose binding protein, glutathione S-transferase). However, the size of these proteins can potentially interfere with the protein to which they are fused. A few short peptides, which are potentially less perturbing, have been used as affinity tags. The most common ones are the 6×histidine tag and the FLAG tag (a 6 amino acid antibody epitope). However, both these affinity tags have disadvantages. The FLAG tag requires the use of an expensive antibody affinity matrix and as a result has not received widespread use. The polyhistidine tag, which binds to metal ions, is very widely used, but requires somewhat harsh conditions (either high concentrations of imidazole or low pH) for elution, which can disrupt macromolecular complexes. In addition, small amounts of metal ions that elute with the protein can inactivate many enzymes. The purity of the eluted protein can be low because many histidine-rich proteins can bind to and elute from metal affinity resins, contaminating the purified protein.
Recently, a fluorescent dye has been developed which specifically interacts with tetracysteine containing helices (Griffin, et al., Science 281:269-272 (1998)). This compound, known as FlAsH (fluorescein arsenical helix binder), has been shown to specifically interact with proteins tagged with a C-C-X
1
-X
2
-C-C containing helix. The interaction is readily reversed by incubation with small dithiols such as ethanedithiol. FlAsH affinity chromatography is a highly specific protein purification method. It is based on the regiospecific interaction of two arsenics in FlAsH with two pairs of cysteines in a target alpha helix. The only requirement for binding is that the protein of interest contain the motif C-C-X
1
-X
2
-C-C within an alpha helix. This motif is rarely found in proteins, and labeling tagged proteins in vivo (Griffin, et al., Science 281:269-272 (1998)) indicate that there are very few eukaryotic proteins that bind to FlAsH. In contrast, many organisms contain histidine-rich proteins, and binding of these proteins to metal ion resins is a major source of contamination.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of a method for protein purification using a modified fluorescein arsenical helix binder (FlAsH) compound, immobilized to a solid support, which can yield substantially pure protein from a single purification step.
A particularly preferred modified bis-arsenical molecule of the invention has the following formula:
The tautomers, anhydrides and salts of the modified bis-arsenical molecule of formula (I) are also included.
Preferably, the modified bis-arsenical molecule specifically reacts with a target sequence, within the bonding partner, to generate a detectable signal, for example, a fluorescent signal.
The modified bis-arsenical molecule preferably is capable of traversing a biological membrane. The modified bis-arsenical molecule preferably includes a detectable group, for example a fluorescent group, luminescent group, phosphorescent group, spin label, photosensitizer, photocleavable moiety, chelating center, heavy atom, radioactive isotope, isotope detectable by nuclear magnetic resonance, paramagnetic atom, and combinations thereof.
In a first aspect, the invention includes a method for isolating a polypeptide of interest including contacting a modified Fluorescein Arsenical Helix (FlAsH) binder compound immobilized on a solid support with a sample containing a polypeptide of interest under conditions that allow binding of the polypeptide to the immobilized FlAsH compound, and eluting the polypeptide of interest, which has been modified by the addition of the FlAsH target sequence motif C-C-X
1
-X
2
-C-C (SEQ ID NO: 1,), where X
1
and X
2
are any amino acid, from the immobilized FlAsH compound. The polypeptide of interest which contains the FlAsH target sequence is the bonding partner of the modified FlAsH compound. “Bonding partner” as used herein refers to a molecule that contains at least the target sequence.
Another aspect of the invention provides a DNA construct containing an origin of replication, a selectable marker, a promoter that allows expression of the polypeptide of interest, and a cloning site, wherein the 5′ end of the cloning site contains a genetically-encoded affinity tag, and wherein the 3′ end of the cloning site contains a FlAsH target sequence motif.
Yet another aspect of the invention provides a DNA construct comprising an origin of replication, a selectable marker, a promoter that allows expression of the polypeptide of interest, and a cloning site, wherein the 5′ end of the cloning site contains a FlAsH target sequence motif, and wherein the 3′ end of the cloning site contains a genetically-encoded affinity tag.
Still another aspect of the invention provides a method for producing a polypeptide of interest which contains at its N-terminus a genetically-encoded affinity tag and at its C-terminus contains a FlAsH target sequence motif including, expressing a DNA sequence which encodes the polypeptide of interest as in the above-mentioned DNA construct, in an appropriate cell type, and producing the polypeptide of interest in an appropriate cell type.
Another aspect of the invention provides a method for isolating a polypeptide of interest, wherein the polypeptide contains at its N-terminus a genetically encoded affinity tag and at its C-terminus a FlAsH sequence motif. The method includes contacting a sample which contains a polypeptide of interest with an affinity resin which binds to the affinity tag; eluting the polypeptides bound to the affinity resin; contacting a modified FlAsH compound immobilized on a solid support with the polypeptides eluted from the first affinity resin, under conditions that allow binding of the polypeptide to the FlAsH comp
Cooke Roger
Matsuka Marija
Naber Nariman
Thorn Kurt
Vale Ronald D.
Gray Cary Ware & Freidenrich LLP
Low Christopher S. F.
Robinson Hope A.
The Regents of the University of California
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