Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-01-28
2001-10-16
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S350000, C536S024300
Reexamination Certificate
active
06303317
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of biological probes, and more specifically to polypeptide probes directed to coiled coil proteins.
BACKGROUND OF THE INVENTION
Interactions among proteins are essential for the controlled function of all living cells, and inappropriate or adventitous protein-protein interactions are the hallmarks of virtually all diseases. Accordingly, biological probes directed at either the proteins themselves, or alternatively at their nucleic acid precursers, are essential for diagnosis of disease and general human health.
Existing biological probes are either nucleic acid-based or amino acid-based. Unfortunately, these prior art biological probes suffer from distinct and somewhat complementary disadvantages. Nucleic-acid based systems include oligonucleotides targeted for specific RNA or DNA sequences, which have the advantages of straightforward synthesis and simple rules for molecular complementarity. However, at present they can only be targeted easily to nucleic acids. Amino acid-based biological probes include antibodies, which can recognize a much broader range of molecules than nucleic acid-based probes. However, they are expensive and time-consuming to produce, requiring purification of the target and maintenance of animals. Moreover, functional antibodies cannot be expressed in intracellular compartments, and their use as biological probes often suffers from varying degrees of antigen expression, non-specific binding and adverse immunogenic reactions.
Accordingly, there is a significant need in the art for a biological probe with broader applications than oligonucleotides and antibodies. Such a probe should combine the advantages of nucleic acid-based probes—quick synthesis, intracellular expression and easy labeling in vitro—with the specificity of antibodies for peptide sequences. Ideally, these probes should be sensitive, stable and recognize a wide range of biological molecules having different biological functions.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to remedy the disadvantages encountered in prior art biological probes, through the provision of heterospecific polypeptide probes directed to coiled coil regions, and novel methods for making and using these probes.
In one embodiment, the present invention contemplates a polypeptide probe having a coiled coil region, wherein the core amino acid residues of the probe coiled coil region have been selected according to the core residue pairing rules so as to favor heterospecific oligomerization with a target coiled coil region. In a further embodiment, the amino acid residues of the probe coiled coil region are selected so as to optimize electrostatic interactions in the edge region of the interhelical interface adjacent to the core. In a specific embodiment, the probe comprises a substantially purified polypeptide sequence (SEQ ID NO: 2) directed to the APC protein. In a further embodiment, the invention comprises an isolated polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2.
In an alternative embodiment, the present invention comprises a coiled coil structure formed between a target coiled coil region and a probe coiled coil region, wherein the amino acid residues located at positions a′ and d′ in the heptads of the probe coiled coil region are selected according to the core residue pairing rules. In a preferred embodiment, a leucine located at position a in a heptad of the target coiled coil region is paired with a preferred amino acid in the a′ position in a heptad of the probe coiled coil region, wherein the preferred amino acid is selected from the group comprising: phenylalanine, isoleucine, lysine, methionine, serine, threonine and valine. In a further embodiment, a leucine located at position d in a heptad of the target coiled coil region is paired with a preferred amino acid in the d′ position in a heptad of the probe coiled coil region, wherein said preferred amino acid is selected from the group comprising tryptophan, valine, arginine, glutamine, phenylalanine, asparagine, methionine, isoleucine and alanine.
In another preferred embodiment, a valine located at position a in a heptad of the target coiled coil region is paired with a preferred amino acid in the a′ position in a heptad of the probe coiled coil region, wherein the preferred amino acid is selected from the group comprising: alanine, isoleucine, leucine, glutamine and serine. In a further embodiment, a valine located at position d in a heptad of the target coiled coil region is paired with a preferred amino acid in the d′ position in a heptad of the probe coiled coil region, wherein said preferred amino acid is selected from the group comprising tyrosine, alanine, leucine, methionine, glutamine and threonine.
In an alternative embodiment, the present invention provides a hetero-oligomer comprising a polypeptide probe and a polypeptide target, wherein the probe comprises a first core region and the target comprises a second core region, and wherein a leucine in an a position in the second core region is paired with a preferred amino acid in an a′ position of the first core region, wherein the preferred amino acid in the a′ position is selected from the group comprising phenylalanine, isoleucine, lysine, methionine, serine, threonine, and valine. In a further embodiment, a leucine in the d position of the second core region is paired with a preferred amino acid in the d′ position of the first core region, wherein the preferred amino acid in the d′ position is selected from the group comprising tryptophan, valine, arginine, glutamine, phenylalanine, asparagine, methionine, isoleucine and alanine.
In an alternative embodiment, the present invention provides a hetero-oligomer comprising a polypeptide probe and a polypeptide target, wherein the probe comprises a first core region and the target comprises a second core region, and wherein a valine in an a position in the second core region is paired with a preferred amino acid in an a′ position of the first core region, wherein the preferred amino acid in the a′ position is selected from the group comprising alanine, isoleucine, leucine, glutamine and serine. In a further embodiment, a valine in the d position of the second core region is paired with a preferred amino acid in the d′ position of the first core region, wherein the preferred amino acid in the d′ position is selected from the group comprising tyrosine, alanine, leucine, methionine, glutamine and threonine.
The present invention also provides a heterospecific polypeptide probe directed to a coiled coil region of a target polypeptide, wherein the melting temperature of the hetero-oligomer formed between the probe and the target at a fixed total protein concentration is higher than the melting temperature of a homo-oligomer formed by either the probe or the target. In a preferred embodiment, the melting temperature of the hetero-oligomer is at least 8° C. higher than the average melting temperature of the homo-oligomers. In an alternative embodiment, the dissociation constant (Kd) of the hetero-oligomer formed between the probe and the target is lower than the dissociation constant of a homo-oligomer formed by either the probe or the target. In a particularly preferred embodiment, the dissociation constant of the hetero-oligomer is equal to or lower than 10 nM.
One embodiment of the methods of the present invention provides a method for making a heterospecific polypeptide probe, comprising the steps of 1) providing a target polypeptide having a target coiled coil region; 2) determining the target polypeptide sequence of the target coiled coil region; and 3) generating a probe polypeptide having a probe coiled coil region, wherein the probe polypeptide sequence of the probe coiled coil region is selected so as to favor heterospecific oligomerization of the probe coiled coil region to the target coiled coil region In a preferred embodiment,
Alber Thomas C.
Allen Victoria
Nautiyal Shivani
Flehr Hohbach Test Albritton & Herbert LLP
Houtteman Scott W.
Lorenz Todd A.
The Regents of the University of California
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