Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-12
2003-12-09
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C436S094000, C530S300000, C530S350000
Reexamination Certificate
active
06660473
ABSTRACT:
BACKGROUND OF THE INVENTION
In general, the invention relates to methods of labeling proteins.
Covalent conjugates of polypeptides with non-peptide “labels” or “tags” form a useful class of reagents in protein research. A conjugate is usually intended to retain the native properties of the protein while gaining a new, non-native property due to the label. Biotinylation, for example, permits proteins to be separated, quantified, or immobilized by mechanisms based on the strong interaction of biotin with avidin or streptavidin (Bayer & Wilchek (1990) Protein Biotinylation, Meth. Enzymol. 184:138-160). Fluorescent or metal-chelating groups can also be introduced to generate newly modified proteins.
It would be convenient to be able to introduce a non-protein label as the protein is produced, but this is only sometimes feasible, for example, if the peptide can be produced by chemical synthesis, and is impractical when the protein is produced biologically. Generally, the conjugate must be formed by treating the peptide with a functional group-specific reagent that contains the label. Moreover, unless the peptide contains only one group attacked by the reagent, this procedure generally yields a mixture of products. This random form of labeling is sometimes adequate, but it is often preferable to modify a protein at a single specified site and to employ the modified product in purified form. For such cases, it would be valuable to have a method of directing the modifying group to a single, preselected location. Such a precisely targeted modification is termed site-directed protein tagging.
SUMMARY OF THE INVENTION
In general, the invention features a protein having a covalently bonded puromycin-tag, the tag being positioned at the C-terminal end of the protein.
In preferred embodiments, the tag is a small molecule (for example, biotin); the tag is a detectable label (for example, fluorescein, rhodamine, or BODIPY, or derivatives thereof); the tag is a functional group (for example, a functional group having a reactivity orthogonal to the reactivity of one of the protein's functional groups); the tag is a tether for attachment to a solid support (for example, a column, bead, or chip); the tag is one member of a specific binding pair; the tag is a phenyl diboronic acid derivative; the puromycin-tag further includes a nucleotide sequence positioned between the tag and the puromycin; and the nucleotide sequence is between about 1-200 nucleotides in length.
In a related aspect, the invention features a method for C-terminal protein tagging, involving (a) providing a nucleic acid sequence encoding the protein; (b) translating the sequence under conditions in which translation stalls at the 3′ end of the sequence, forming a stalled translation complex; and (c) contacting the stalled translation complex with a puromycin-tag under conditions in which the puromycin-tag is covalently bonded to the C-terminus of the protein.
In preferred embodiments, the tag is attached to the 5′-hydroxy group of puromycin; the tag is attached to the 5′-hydroxy group of the puromycin through a phosphate group; the nucleic acid sequence encoding the protein contains no stop codons; the translation step is carried out in the substantial absence of at least one translation release factor; the 3′-end of the nucleic acid sequence encoding the protein is covalently linked to a DNA oligomer; the tag is a small molecule (for example, biotin); the tag is a detectable label (for example, fluorescein, rhodamine, or BODIPY, or a derivative thereof); the tag is a functional group; the protein has a first functional group and the tag is a second functional group, wherein the first functional group has a reactivity orthogonal to the reactivity of the second functional group; the tag is a tether for attachment to a solid support (for example, a column, bead, or chip); the tag is one member of a specific binding pair; the tag is a phenyl diboronic acid derivative; the puromycin-tag further includes a nucleotide sequence positioned between the tag and the puromycin; and the nucleotide sequence is between about 1-200 nucleotides in length.
By a “protein” is meant any two or more naturally occurring or modified amino acids joined by one or more peptide bonds. “Protein,” “peptide,” and “polypeptide” are used interchangeably herein.
By a “puromycin-tag” is meant puromycin having a covalently bonded structural or functional moiety which is not native to the puromycin molecule and which is chosen from the group consisting of a detectable label, a chemically reactive functional group, a small molecule, a protein or peptide, a peptoid, a naturally occurring or non-naturally occurring polymer, a solid-phase bound tether, a carbohydrate, or a nucleic acid (preferably, of between about 1-200 nucleotides) which does not encode the protein to which the puromycin-tag is itself covalently linked. By a “nucleic acid” is meant any two or more covalently bonded, naturally occurring or modified nucleotides and includes DNA, RNA, and PNA. Preferred puromycin-nucleic acid tags include 5′-C-C-puromycin-3′.
By a “small molecule” is meant a molecule having a molecular weight of approximately 2000 Daltons or less, preferably, 1500 Daltons or less, more preferably, 1000 Daltons or less, and, most preferably, 500 Daltons or less.
By a “functional group” is meant any moiety of, or arrangement of atoms in, a molecule which exhibit some chemical reactivity.
The present invention provides a number of advantages over current chemical and enzymatic protein-tagging methods. For example, the tag is introduced in the final step of translation on the ribosome. This modification is advantageous because tagged proteins may be generated in a single preparative step. In addition, the tag is introduced in translation buffer under conditions which enhance protein stability. Again, this provides for increased product yield and optimized protein quality. In particular, although several schemes for N-terminal (Drijfhout et al. (1990) Anal. Biochem. 187: 349-354; Wetzel et al. (1990) Bioconjugate Chem. 1: 114-122) and C-terminal (Schwarz et al. (1990) Meth. Enzymol. 184: 160-162; Rose et al. (1989) Peptides 1988 (G. Jung and E. Bayer, eds.) pp. 274-276, Walter de Gruyter & Co., New York) tagging have been described, many of these methods involve a tagging step that is carried out under conditions which disrupt protein structure. For example, modification at non-physiological pH and temperature or in the presence of non-aqueous solvents or chemicals, followed by purification of the modified protein, disrupts folding thus leading to a non-functional product. In contrast, the present tagging method is performed under native conditions. Finally, in yet another advantage, the present invention enables the introduction of a tag regioselectively at the C-terminus of a protein, facilitating the production of native proteins carrying desired C-terminal structural or functional elements in a simple and efficient way.
The C-terminally tagged polypeptides and proteins produced by the methods of the present invention may be used in any appropriate technique, for example, in any affinity purification method, protein detection method (for example, using proteins having C-terminal fluorescein tags), structure function or protein dynamics analyses (for example, using proteins having C-terminal reporter tags), pharmaceutical analyses (for example, using proteins having detectable C-terminal tags which allow for a determination of cellular protein uptake or cellular localization), or protein display technology (for example, using solid phase tags to generate protein arrays on microchips).
Other features and advantages will be apparent from the following detailed description and from the claims.
REFERENCES:
patent: 5723584 (1998-03-01), Schatz
patent: 6228994 (2001-05-01), Yanagawa et al.
patent: 98-320093 (2000-05-01), None
patent: WO 98/31700 (1998-07-01), None
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patent: WO 0
Kuimelis Robert G.
Lohse Peter
McPherson Michael
Clark & Elbing LLP
Lu Frank
Phylos, Inc.
Whisenant Ethan
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