Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Utility Patent
1998-03-16
2001-01-02
Kunz, Gary L. (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S069100, C435S173300, C530S350000
Utility Patent
active
06169172
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to polypeptides which enable proteins which are produced in eukaryotic cells to be secreted directly.
In recent years, the baculovirus/insect cell system has been used successfully for expressing various genes of interest. In this system, as, in a more general manner, in the different systems which are known for producing recombinant proteins in host cells, it is desirable to have available means for exporting the said recombinant proteins into the medium in which the cells are cultured. This is because exporting these proteins makes it easier to harvest and purify them.
Generally, the approach is to attempt to use the standard secretion pathway, which involves the endoplasmic reticulum and the Golgi apparatus, in order to achieve the export of a recombinant protein from a eukaryotic host cell.
To this end, a cleavable N-terminal signal peptide is added to the protein which it is desired to have secreted by fusing a sequence encoding the said signal peptide to the sequence encoding the recombinant protein. The presence of this signal peptide enables the polypeptide to be translocated across the membrane of the endoplasmic reticulum, by way of which the polypeptide will be transported, in the vesicles of the Golgi apparatus, right up to the plasma membrane. However, this approach does not always enable the hoped-for result to be achieved [JARVIS et al., J. Biol. Chem. No. 268, pp. 16754-16762, (1993)].
The principal drawback of this strategy resides in the fact that passage by way of the endoplasmic reticulum and the Golgi apparatus can entail the addition, onto the polypeptide, of sugar residues which can result in the protein being folded incorrectly and/or in new epitopes being formed.
Moreover, it was observed, during the course of earlier work which was carried out by the inventors' group regarding expression of the prolactin receptor, or different portions of this receptor, in recombinant baculoviruses [CAHOREAU et al., Biochimie, No. 74, pp. 1053-1065, (1992)], that, when the intracytoplasmic region of the prolactin receptor was expressed on its own, it was excreted into the culture medium even though it did not possess any signal peptide.
Subsequent studies on the structure of this intracytoplasmic region showed that it was not N-glycosylated (even though it possesses several potential glycosylation sites) and that it was ubiquitinylated [CAHOREAU et al., FEBS Letters, No. 350, pp. 130-234, (1994)].
The prolactin (PRL) receptor belongs to a family of receptors (cytokine receptors) which also includes the receptors for growth hormone (GH) and those for several interleukins, those for erythropoietin and for GM-CSF (=granulocyte-macrophage colony stimulating factor) and the receptor for interferon &ggr;, etc. These receptors consist of a N-terminal extracellular region, of a transmembrane region and of a C-terminal intracytoplasmic region (for review, see, for example [DUSANTER-FOURT et al., M{acute over (e)}decine/Sciences Synth{grave over (e)}se, 10, p. 825-835, (1994)]).
The prolactin receptor exists in two forms: a short form, which is composed of 291 amino acids, and a long form, which consists of 591 amino acids in the rat, 592 amino acids in the rabbit and 598 amino acids in man.
The growth hormone receptor consists of 620 amino acids (in man and rabbit) and exhibits a large number of regions which are homologous with the long form of the prolactin receptor [KELLY et al., in Recent Progress in Hormone Research, 48, p. 123 (Academic Press Ed. (1993); KELLY and DJIANE, U.S. Pat. No. 4,992,398]: in particular, 4 regions which are conserved are found in the intracytoplasmic region.
The other receptors of the same family (receptor for erythropoietin, receptors for growth factors, receptors for interleukins 2, 3, 4, 5, 6, 7, and 9, GM-CSF receptor, G-CSF receptor, receptor for interferon &ggr;, etc.) also possess very closely related structures and the domains of different receptors can be combined with each other in order to produce functional chimeric receptors which comprise the extracytoplasmic domain of a first receptor and the intracytoplasmic domain of a second receptor, which domains are separated by a transmembrane domain belonging either to the first or to the second receptor [MOORE et al., FASEB J., 9 (6), A1414, (1995); CIOFFI et al., FASEB J., 7 (3-4), A430, (1993); DUSANTER et al., Journal of Cellular Biochemistry Supplement, 18B, p. 276, (1994)].
The inventors then showed that the intracytoplasmic region of the prolactin receptor was not only capable of being exported into the culture medium when it was expressed on its own, but was also capable of enabling a protein (termed “passenger” protein) to be exported when this protein was fused to its C-terminal end. The inventors additionally defined the minimum region which is responsible for this property.
Moreover, the inventors also observed that, when expressed in a baculovirus/insect cell system, the intracytoplasmic region of the growth hormone (GH) receptor was excreted into the culture medium in a manner which was analogous to that in which the intracytoplasmic region of the prolactin receptor was excreted. Thus, the inventors identified, in particular, a polypeptide of 372 amino acids which is encoded by the fragment of the sequence encoding the growth hormone receptor which is located between the NcoI site and the stop codon of the said sequence, and a polypeptide of 329 amino acids which is encoded by the fragment of the sequence encoding the growth hormone receptor which is located between the ClaI site and the stop codon of the said sequence.
The demonstration, by the inventors, of these characteristics of the receptors for prolactin and for growth hormone make it possible to envisage using the intracytoplasmic domains of cytokine receptors, and more specifically the intracytoplasmic domains of the receptors for prolactin and growth hormone, as transporters for enabling proteins of interest, whose localization is normally cytoplasmic or nuclear, to be exported from a host cell.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to the use of a protein whose polypeptide sequence is selected from the group consisting of:
the sequence of the intracytoplasmic domain of the prolactin receptor, namely the region consisting of amino acids 235 to 591, in accordance with the numeration of the amino acids described by KELLY et al. (1993, loc. cit.), of the long form of the said receptor in the rat (SEQ ID NO:6) of amino acids 235 to 598 of the long form of the said receptor in man (SEQ ID NO:2), and of amino acids 235 to 592 of the long form of the said receptor in the rabbit (SEQ ID NO:4));
the sequence of the fragment of the prolactin receptor consisting of amino acids 240 to 592 of the long form of the said receptor in the rabbit (SEQ ID NO:4), of amino acids 235 to 591 of the long form of the said receptor in the rat (SEQ ID NO:6), and of amino acids 235 to 598 of the long form of the said receptor in man (SEQ ID NO:2);
the sequence of the intracytoplasmic domain of the growth hormone (GH) receptor, consisting, in man, of the C-terminal region, of 350 amino acids, of this receptor (SEQ ID NO:1);
the sequence of a fragment of the growth hormone (GH) receptor, comprising all or part of the intracytoplasmic domain of the said receptor, and comprising amino acids 271 to 620 of the said receptor in man (SEQ ID NO:1) (for example the fragment consisting of amino acids 249 to 620 (SEQ ID NO:1)), or else amino acids 272 to 620 of the said receptor in the rat (SEQ ID NO:5), or else amino acids 271 to 620 of the said receptor in the rabbit (SEQ ID NO:3), in order to achieve the secretion of a protein of interest which is produced in a eukaryotic host cell, in particular an insect host cell.
In order to implement the present invention, a eukaryotic cell expression system is caused to produce a chimeric fusion protein which comprises the protein of interest, which it is desired to expor
Cahoreau Claire
Cerutti Martine
Devauchelle G{acute over (e)}rard
Garnier Laurence
Basi Nirmal S.
Institut National de la Recherche Agronomique (INRA)
Kunz Gary L.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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