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
2000-02-29
2004-03-30
Ulm, John (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...
C435S005000, C435S252300, C435S320100, C536S023740
Reexamination Certificate
active
06713278
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the production of membrane receptors in a baculovirus/insect cell system.
BACKGROUND OF THE INVENTION
Over the past few years, heterologous expression systems have often been used to study the expression as well as the pharmacological and biochemical characteristics of membrane receptors.
Although a significant expression can be obtained in some expression systems in mammalian cells, there have been problems, in particular in the case of some types of receptors such as the G-protein-coupled receptors.
The G-protein-coupled receptors belong to the superfamily of receptors with seven transmembrane domains. They comprise, for example, the adrenergic or muscarinic receptors, and all have the same structure which is made up of a polypeptide chain comprising seven hydrophobic domains which cross the membrane lipid bilayer.
When it is desired to express these receptors in mammalian cell systems, a relatively low density of receptors expressed by the said cells, rarely exceeding a few picomoles of receptor per milligram of membrane protein, is generally obtained. Although these levels of expression are sufficient for a functional and pharmacological characterization, they clearly limit the type of biochemical, biophysical and structural studies which can be carried out. A fortiori, this expression system cannot be used for the production of receptors in a large quantity, for example for their therapeutic use.
To increase the quantity of receptors obtained, various teams have sought to produce them in a baculovirus/insect cell system; in many cases, baculoviruses expressing G-protein-coupled receptors have been able to produce these recombinant receptors in cells of the
Spodoptera frugiperda
Sf9 or Sf21 lines, up to levels reaching 30 to 100 picomoles per milligram of membrane protein. These systems have made it possible to make significant progress in the study of the palmitoylation of receptors and also to study the effects produced by various agonists and antagonists, or to carry out the reconstitution of artificial receptors.
However, the baculovirus/insect cell system has the major disadvantage of expressing a high proportion of inactive receptors. The receptors, which are recovered in the membrane fraction of the cells infected with the baculoviruses are in an immature and incompletely glycosylated form. This probably results from a saturation of the normal post-translational maturation pathway, which brings about the retention of immature receptors in the membranes of the endoplasmic reticulum or in the Golgi apparatus. To obtain functional receptors, it is necessary in this case to include a purification step based on the biological activity of the receptor (for example an affinity chromatography step).
It would therefore be necessary to develop a system which makes it possible to easily separate the plasma membrane comprising the mature receptors from the other membrane fractions such as the endoplasmic reticulum or the membranes of the Golgi apparatus which comprise the biologically inactive, immature receptor.
SUMMARY OF THE INVENTION
It has recently been shown that the infection of Sf9 cells with a baculovirus encoding the HIV1 Gag gene (Pr55 Gag) brings about the budding of particles carrying the Gag protein (Gag particles) which are released into the extracellular medium. It has been suggested that these Gag particles carry with them, during their budding, the plasma membrane and the proteins associated with it.
The inventors have formulated the hypothesis that the coexpression, in a baculovirus/insect cell system, of a G-protein-coupled receptor and of Pr55 Gag can promote the release of the Gag particles expressing only the mature receptors which are correctly inserted into the plasma membrane. To test this hypothesis, the inventors infected Sf9 cells with baculoviruses encoding the human adrenergic receptor &bgr;2AR and the Pr55 Gag protein. Surprisingly, they then observed that the &bgr;2AR receptor is almost completely absent from the Gag particles, but is, on the other hand, present at a high density in extracellular baculovirus particles. In addition, the receptors expressed in these extracellular baculoviruses are correctly glycosylated and normally active.
The present invention relates to the use of these extracellular baculoviruses for the production of preparations of a membrane receptor.
The subject of the present invention is a method of producing a recombinant membrane receptor in a baculovirus/insect cell system, from a culture of insect cells infected with a recombinant baculovirus expressing the gene encoding the membrane receptor, which method is characterized in that the membrane receptor is obtained from extracellular baculoviruses produced by the infected cells.
According to a preferred embodiment of the present invention, the receptor belongs to the superfamily of receptors with seven transmembrane domains; this is for example a receptor of the family of G-protein-coupled receptors.
Recombinant baculoviruses expressing the gene encoding the membrane receptor which it is desired to produce are obtained by cloning the gene under transcriptional control of an appropriate promoter of the baculovirus, according to methods well known per se to persons skilled in the art.
Any strong baculovirus promoter which can be used for the expression of heterologous genes, such as for example the polyhedrin promoter (polh) or that of the P10 protein, may be used for the production of a recombinant baculovirus which can be used within the framework of the present invention.
According to a preferred embodiment of the method in accordance with the invention, it comprises a step during which the extracellular baculoviruses produced by the infected cells are harvested and they are separated from the cellular fractions. The harvesting and the separation of the extracellular baculoviruses may be carried out by successive centrifugations, for example in the following manner: a first centrifugation is carried out at about 500×g, at the end of which the supernatant containing the extracellular baculoviruses is collected. This supernatant is subjected to a centrifugation at about 45,000×g; the resulting pellet which contains the extracellular baculoviruses is resuspended, and the suspension is subjected to a centrifugation at about 500×g; the supernatant resulting from this centrifugation is centrifuged at about 45,000×g, and the pellet, which contains the extracellular baculoviruses, is recovered. Advantageously, the extracellular baculoviruses may also be purified by sucrose gradient centrifugation, or any other equivalent method.
According to another preferred embodiment of the method in accordance with the invention, it comprises a step during which the extracellular baculoviruses produced by the infected cells are lysed; advantageously, it also comprises a step during which the lysate obtained at the end of the preceding step is fractionated, and the fraction comprising the membrane receptor is recovered.
The purified preparations and the lysates of extracellular baculoviruses, as well as their fractions comprising the membrane receptor, which are capable of being obtained by the methods defined above constitute membrane receptor preparations which also form part of the subject of the present invention. These preparations consist of active and fully mature receptors, unlike the membrane receptor preparations obtained in the prior art from the plasma membranes of infected cells, which comprise a high proportion of inactive receptors, and which can only be used after an additional step of purification on the basis of the activity of the relevant receptors, for example after affinity chromatography.
By contrast, the membrane receptor preparations in accordance with the invention are characterized in that, prior to any purification carried out on the basis of the activity of the relevant receptor, at least 90%, and preferably at least 95%, of the receptor is in an active form.
Preparations, in accordance
Boulanger Pierre
Bouvier Michel
Loisel Thomas
Marullo Stefano
Strosberg Arthur Donny
Nixon & Vanderhye
Ulm John
Valorisation-Recherche, Limited Partnership
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