Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1995-04-13
2003-11-11
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S254210, C435S254220, C435S254300
Reexamination Certificate
active
06645755
ABSTRACT:
The invention relates to a vector which can be replicated in cultures of unicellular organisms, this vector containing a gene coding for a eucaryotic protein having the biological activity of a membrane receptor and interacting with a regulatory protein—called G protein—capable of binding molecules of guanosine triphosphate (GTP).
The invention also relates to unicellular organisms transformed by genes coding for these membrane receptors and bearing at their surface peptide sequences recognized by specific ligands of the membrane receptors.
The invention also relates to kits for the identification of mammalian proteins which have properties making it possible to verify whether they belong to a family of mammalian membrane receptors or, conversely, to study the degree of affinity of certain substances for specific membrane receptors.
Generally speaking, the G proteins are proteins having the capacity to interpose themselves structurally and functionally between receptors and enzymes catalyzing the production of intracellular mediators (such as adenylate cyclase, guanylate cyclase, the phospholipases, the kinases) or between receptors and ionic channels, the controlled opening of which leads to a flux of ions (such as calcium, potassium, sodium and hydrogen ions) into the cell.
These proteins have transduction and coupling functions and are characterized by a monomeric structure composed of three protein subunits alpha, beta, gamma. Different G proteins can be distinguished on the basis of variations observed in their alpha subunit and sometimes in their beta subunit. These G proteins have been described in detail in the article by Allen M. Spiegel (21).
Membrane receptors of mammalian cells normally coupled to a G protein have been identified by cloning, supplemented by the analysis of their sequence in the case of some of them. These receptors include regions exposed at the surface of the host cells which have specific affinities for a large variety of ligands, hormones, neurotransmitters and other sensory stimulants capable of transmitting signals to the interior of these cells via the corresponding receptors and the G proteins.
Receptors of interest in the context of the invention are, in particular, the following receptors:
&bgr;-adrenergic receptors: (&bgr;1 avian receptor, &bgr;1 and &bgr;2 mammalian receptors), receptors for glucagon, the adrenocorticotropic hormone, the parathyroid hormone (PTH), the thyroid-stimulating hormone, the growth hormone releasing factor, prostaglandin E, D1 dopaminergic receptors, the intestinal vasoactive peptide receptor, the mucarinic receptors M1 to M6, the S1 and S2 serotonin receptors, the D2 dopaminergic receptor, the receptors for the neuropeptide substance K, vasopressin, somatostatin, bombesin, cholecystokinin (CCK), light receptors, the receptor for the chemotactic peptide Met-Leu-Phe, the &agr; adrenergic receptors (&agr;1, &agr;2), the H1 histamine receptor, the opioid receptor (enkephalins, endorphins), the receptors for the neuropeptide substance P, angiotensin II, prostaglandin, the corticotropic hormone releasing factor, bradykinin, secretin and the olfactory receptors.
A preferred family of receptors according to the invention which possess genetic and molecular properties which are essentially common to all of them, comprises &bgr;1, &bgr;2, &agr;1 and &agr;2-adrenergic receptors (1) (2) (3) (4), muscarinic receptors belonging to the subtypes M1 to M4 (5) (6) and the receptor for the neuropeptide known as “substance K” (7). These receptors appear to possess a common structural organization within the plasma membrane, usually characterized by seven hydrophobic transmembrane segments between which are inserted extra- and intra-cellular loops, by an extracellular amino-terminal region and by a cytoplasmic carboxyl-terminal region.
Several genes coding respectively for some of these receptors have been made capable of expressing themselves after their introduction into cultures of mammalian cells belonging to foreign species. The operations of transformation necessary for the expression of these genes in mammalian cells belonging to foreign species are quite complex and cannot easily be discussed in terms of the expression of other receptors.
The aim of the invention is to furnish procedures and agents making it easy to identify and study new membrane receptors exhibiting structural characteristics in common with the membrane receptors of the type indicated above. Conversely, the aim of the invention is to facilitate the study of the possible degree of affinity of molecules capable of behaving as ligands with respect to these membrane receptors.
In particular, the invention relates to a vector, which can replicate in a culture of a host cell, containing a nucleotide insert coding for a sequence of amino acids contained in a mammalian protein and having the biological activity of a membrane receptor, this vector being characterized in that the insert is placed under the control of a sequence included in this vector allowing the expression of this insert in the host cell, and in particular including a promoter recognized by the polymerases of this unicellular host, and in that this insert codes for a sequence of amino acids contained in a protein possessing structural elements in common with the above-mentioned membrane receptors, in particular the &agr;- or &bgr;-adrenergic receptors, the muscarinic receptors, the neuropeptide receptors, in particular for “substance K”.
The invention is the result of the discovery that placing a sequence of nucleotides coding for a mammalian membrane receptor under the control of a promoter recognized by the polymerases of unicellular host under conditions which allow the expression of this sequence of nucleotides within the unicellular organism also makes possible, when this expression occurs, the transport of the expressed product into the membranes of the unicellular host, and that this expression is also accompanied by the exposure, also at the exterior of these unicellular hosts, of sites characteristic of the membrane receptors which appear at the surface of the cells of mammalian origin, and do so in a spatial configuration that is conserved at the molecular level such that these sites can be recognized by the same ligands.
By “unicellular host” is understood any organisms capable of being maintained in culture in the absence of cellular differentiation. The unicellular organism of choice is constituted by
Escherichia coli
. However, other microorganisms can be used with equal facility, provided of course that vectors are known for each of them, especially of the plasmid type, which are capable of self replication and nucleotide sequences which can be inserted into these vectors and which are capable, when they are followed in these vectors by an insert coding for a polypeptide having the properties of a membrane receptor under the conditions indicated above, of insuring the expression of this insert in the selected organisms and its transport into the membranes of these unicellular hosts.
In the case where the expression is to be carried out in
E. coli
, the sequence directing the expression of the insert may include, for example, the elements of regulation, in particular the promoter of the lactose operon or the tryptophan operon.
Other unicellular microorganisms capable of being used and, consequently, other vectors which can themselves be modified by a sequence capable of directing the expression of a membrane receptor or similar structure in these unicellular organisms are indicated in the table below:
Unicellular
Vector
Promoter
Organism
type
region
S. Cerevisiae
plasmid 2&mgr;
alcohol
dehydrogenase I
(ADH. I)
S. Cerevisiae
plasmid PBM 258
galactokinase
promoter gene
(GAL I)
The above-mentioned organisms and the corresponding, modified vectors advantageously lend themselves to the expression of genetic sequences coding for molecules having structural properties in common with mammalian membrane receptors already known, and to their transport to the membrane of these unicellular h
Delavier Colette
Emorine Laurent
Marullo Stefano
Strosberg Donny
Centre National de la Recherche Scientifique
Eyler Yvonne
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
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