Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-16
2004-08-31
Kunz, Gary (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S320100, C435S325000, C530S350000, C536S023500
Reexamination Certificate
active
06783952
ABSTRACT:
BACKGROUND OF THE INVENTION
Voltage-dependent Ca
2+
channels (VDCCs) are heteromultimeric complexes present in both neuronal and non-neuronal tissues, including heart and skeletal muscle. VDCCs are minimally composed of three subunits: a pore-forming transmembrane &agr;
1
subunit, a hydrophilic intracellular &bgr; subunit, and a membrane-associated &agr;
2
&dgr; subunit; a transmembrane &ggr; subunit is also found in skeletal muscle tissue. Multiple subtypes and/or splice variants of the &agr;
1
, &bgr;, and &agr;
2
&dgr; subunits have been found.
Gabapentin ((1-aminomethyl)cyclohexane acetic acid or Neurontin) is a structural analogue of GABA, which is mainly used as an adjunctive therapy for epilepsy. Recent research suggests that gabapentin may also have clinical utility for various indications including anxiety and pain. Although designed as a lipophilic GABA-mimetic, gabapentin does not have a high affinity for either GABA
A
or GABA
B
receptors, GABA uptake sites, or the GABA-degrading enzyme GABA-transaminase (EC 2.6.1.19).
A novel high affinity binding site for [
3
H]gabapentin in rat, mouse, and porcin brains has been characterized. Recently, the [
3
H]gabapentin-binding protein was isolated from pig brain and identified as the &agr;
2
&dgr;-1 subunit of VDCCs. None of the prototypic anticonvulsant drugs displace [
3
H]gabapentin binding from the &agr;
2
&dgr;-1 subunit. [
3
H]Gabapentin-binding is stereospecifically inhibited by two enantiomers of 3-isobutyl GABA. The rank order of potency of gabapentin, and S- and R-isobutyl GABA, at the [
3
H]gabapentin binding site mirrors their anticonvulsant activity in mice. However, electrophysiological studies have yielded conflicting data on the action of gabapentin at VDCCs.
The &agr;
2
&dgr; subunit is derived from a single gene, the product of which is extensively post-translationally modified particularly through the cleavage of the signal sequence. The polypeptide is cleaved to form disulfide-bridged &agr;
2
and &dgr; peptides, both of which are heavily glycosylated. Although it seems clear today that the &agr;
2
and &dgr; peptides are membrane-associated peptides, it is unclear whether these peptides comprise one or several transmembrane domains. Furthermore, the location, size and structural configuration of these eventual transmembrane domains remains to be determined.
But in any event, the fact that &agr;
2
&dgr; is a membrane-associated protein, regardless of its precise structural configuration, renders its large scale expression in recombinant systems difficult. Indeed, as the &agr;
2
&dgr; protein is targeted to the membrane, it requires detergent solubilisation to release it for purification. This important drawback imposes considerable restrictions for any potential applications requiring large amounts of recombinant protein. Furthermore, the various subtypes of &agr;
2
&dgr; subunits are different proteins with very low homologies. It is therefore extremely difficult to predict their respective behaviors, for example in gene truncation experiments.
The only assay currently available for the screening of ligands that bind the &agr;
2
&dgr; subunit involves the use of pig membrane extracts as a source of the &agr;
2
&dgr; subunit. Such an assay presents major inconvenients. Firstly, because the assay material is a membrane extract, it is very difficult to accurately determine the protein composition from one assay preparation to another particularly with regard to the subtype. Also, the presence of various impurities in the assay preparation is a problem in small plate assays. Furthermore, as the protein preparation lacks homogeneity, the interaction between the targeted protein and the assay plate is often quite uneven. This renders the streamlining of the assay in a high throughput format almost impossible to achieve.
SUMMARY OF THE INVENTION
In the context of the present invention, the inventors have found that it was possible to delete a portion of the nucleotide sequence encoding a eukaryotic, preferably a mammal cerebral cortical voltage-dependent calcium channel &agr;
2
subunit to yield a soluble secreted protein which retains its affinity for [
3
H]gabapentin.
The Invention Concerns:
1) A purified or isolated nucleic acid encoding a mammalian secreted soluble cerebral cortical voltage-dependent calcium channel &agr;
2
&dgr;-2, &agr;
2
&dgr;-3 or &agr;
2
&dgr;-4 subunit polypeptide.
2) A purified or isolated nucleic acid according to 1), comprising a polynucleotide having at least 90% identity with the sequence encoding:
from amino acid 1 to between amino-acids 1027 and 1062 of SEQ ID NO:20 for &agr;
2
&dgr;-2,
from amino acid 1 to between amino-acids 984 and 1019 of SEQ ID NO:22 for &agr;
2
&dgr;-3.
3) A purified or isolated nucleic acid according to 1), having at least 90% identity with the sequence encoding:
from amino acid 1 to between amino-acids 1047 and 1062 of SEQ ID NO:20 for &agr;
2
&dgr;-2,
from amino acid 1 to between amino-acids 1004 and 1019 of SEQ ID NO:22 for &agr;
2
&dgr;-3.
4) A purified or isolated nucleotide sequence according to 1) wherein said sequence is the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19, or SEQ ID NO:21.
5) A purified or isolated nucleic acid, having at least 90% identity with the nucleotide sequence of SEQ ID NO:19 or SEQ ID NO:21.
6) A purified or isolated polynucleotide comprising at least 10 consecutive nucleotides of the nucleotide sequence of SEQ ID NO:19 or SEQ ID NO:21.
7) A polynucleotide probe or primer hybridizing, under stringent conditions, with the nucleotide sequence of SEQ ID NO:19 or SEQ ID NO:21.
8) A method for the amplification of a nucleic acid encoding a mammalian secreted soluble cerebral cortical voltage-dependent calcium channel &agr;
2
&dgr;-n subunit polypeptide wherein n is 2, 3 or 4, said method comprising the steps of:
(a) contacting a test sample suspected of containing the target secreted soluble &agr;
2
&dgr;-n subunit nucleic acid, or a sequence complementary thereto, with an amplification reaction reagent comprising a pair of amplification primers located on either side of the &agr;
2
&dgr;-n subunit nucleic acid region to be amplified, and
(b) optionally, detecting the amplification products.
9) A kit for the amplification of a nucleic acid encoding a secreted soluble &agr;
2
&dgr;-n subunit polypeptide wherein n is 2, 3 or 4, or a complementary sequence thereto in a test sample, wherein said kit comprises:
(a) a pair of oligonucleotide primers which can hybridize, under stringent conditions, to the secreted soluble &agr;
2
&dgr;-n subunit nucleic acid region to be amplified;
(b) optionally, the reagents necessary for performing the amplification reaction.
10) A recombinant vector comprising a nucleic acid according to 1).
11) A recombinant host cell comprising a nucleic acid according to 1).
12) A method for producing a secreted soluble &agr;
2
&dgr;-n subunit wherein n is 2, 3 or 4, and said method comprises the steps of:
(a) inserting the nucleic acid encoding the desired &agr;
2
&dgr;-n subunit polypeptide in an appropriate vector;
(b) culturing, in an appropriate culture medium, a host cell previously transformed or transfected with the recombinant vector of step (a);
(c) harvesting the culture medium thus obtained or lyse the host cell, for example by sonication or osmotic shock;
(d) separating or purifying, from said culture medium, or from the pellet of the resultant host cell lysate, the thus produced &agr;
2
&dgr;-n subunit polypeptide of interest.
13) A purified or isolated recombinant polypeptide comprising the amino acid sequence of a secreted soluble &agr;
2
&dgr;-2, &agr;
2
&dgr;-3 or &agr;
2
&dgr;-4 subunit polypeptide.
14) A recombinant polypeptide according to 13), having at least 80% amino-acid identity with a polypeptide comprising:
from amino acid 1 to between amino acids 1027 and 1062 of the amino acid sequence of SEQ ID NO:20, or
from amin
Bertelli François
Brown Jason Peter
Ashbrook Charles W.
Baude Eric
Kunz Gary
Kurlandsky David R.
Murphy Joseph F.
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