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Start Over Author "V Bozon" Journal journal of molecular endocrinology
3 results on '"V Bozon"'

1. High-level expression of recombinant porcine LH receptor in baculovirus-infected insect cells or caterpillars

Author

J-J Remy, M. Severini, G. Biache, Edith Pajot-Augy, J-C Pernoller, J-C Huer, V. Bozon, Laurence Couture, Roland Salesse, Unité de biologie cellulaire et moléculaire, Institut National de la Recherche Agronomique (INRA), Unité de lutte biologique, Laboratoire d'études des protéines, Lutte Biologique (ULB), and Laboratoire de biologie de la rhizosphère

Subjects
LH, Swine, Molecular Sequence Data, Gene Expression, Spodoptera, Cell Line, law.invention, 03 medical and health sciences, Endocrinology, Multiplicity of infection, Cell surface receptor, law, Animals, Secretion, Amino Acid Sequence, Cloning, Molecular, Receptor, Molecular Biology, 030304 developmental biology, 0303 health sciences, Chemistry, 030302 biochemistry & molecular biology, luteinizing hormone/choriogonadotropin receptor, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Receptors, LH, Ligand (biochemistry), Molecular biology, Recombinant Proteins, Lepidoptera, Kinetics, Ectodomain, Recombinant DNA, Baculoviridae, Subcellular Fractions
Abstract

Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 107 receptors/cell or 3 μg active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0·6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.

Published
1995
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2. Peptide and immunochemical mapping of the ectodomain of the porcine LH receptor.

Author

Couture L, Naharisoa H, Grebert D, Remy JJ, Pajot-Augy E, Bozon V, Haertle T, and Salesse R

Subjects
Amino Acid Sequence, Animals, Antibodies pharmacology, CHO Cells, Consensus Sequence, Cricetinae, Cyclic AMP metabolism, Enzyme-Linked Immunosorbent Assay, Exons, Kinetics, Luteinizing Hormone pharmacology, Models, Structural, Molecular Sequence Data, Peptide Fragments chemical synthesis, Peptide Fragments immunology, Peptide Fragments pharmacology, Rats, Receptors, LH biosynthesis, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Signal Transduction drug effects, Swine, Transfection, Luteinizing Hormone metabolism, Protein Structure, Secondary, Receptors, LH chemistry, Receptors, LH metabolism
Abstract

The LH/hCG receptor is a G protein-coupled receptor with an N-terminal extracellular domain involved in hormone-receptor interaction. The recombinant porcine receptor, stably expressed in Chinese hamster ovary (CHO) cells, has the same characteristics (Kd and cAMP production) as in Leydig cells. Six synthetic peptides derived from the receptor ectodomain and two polyclonal anti-peptide sera were tested in the homologous system porcine LH and porcine LH receptor. Their ability to inhibit hormone binding and signal transduction on CHO cells expressing the recombinant receptor was evaluated. Peptides 25-40 and 107-121 exhibited a high transduction inhibition as compared with hormone binding, peptides 21-36, 102-111, and 102-121 inhibited hormone binding more efficiently than signal transduction, and peptide 7-24 exhibited inhibition of both hormone binding and hormone-induced cAMP production. Immunoglobulins against peptides 21-36 and 102-111 inhibited both hormone binding and receptor activation suggesting that these sequences are located on the receptor surface. The data suggest that multiple, discontinuous regions of the extracellular domain of porcine LH receptor are involved in hormone binding and signal transduction. Two minimum critical sequences, 21-24 and 102-107, are involved in hormone binding and vicinal segments may be implicated in signal transduction.

Published
1996
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3. Influence of promoter and signal peptide on the expression and secretion of recombinant porcine LH extracellular domain in baculovirus/lepidopteran cells or the caterpillar system.

Author

Bozon V, Remy JJ, Pajot-Augy E, Couture L, Biache G, Severini M, and Salesse R

Subjects
Animals, Base Sequence, Cell Line, Kinetics, Larva, Luteinizing Hormone genetics, Luteinizing Hormone metabolism, Molecular Sequence Data, Moths cytology, Moths growth & development, Peptide Fragments biosynthesis, Peptide Fragments metabolism, Recombinant Fusion Proteins metabolism, Spodoptera cytology, Spodoptera growth & development, Spodoptera metabolism, Swine, Genetic Vectors, Luteinizing Hormone biosynthesis, Melitten genetics, Moths metabolism, Nucleopolyhedroviruses genetics, Peptide Fragments genetics, Promoter Regions, Genetic, Protein Sorting Signals physiology, Recombinant Fusion Proteins biosynthesis
Abstract

Overexpression of the porcine LH receptor (pLHR) ectodomain has been achieved using the baculovirus-insect cell system but mostly in an aggregated form with no secretion. In order to carry this out, new baculoviruses were selected to produce the pLHR ectodomain in insect Sf9 cells and caterpillars. In pLHR-P10-297 and pLHR-mel-319 baculoviruses, pLHR cDNA was under the control of the P10 promoter and the polyhedrin gene promoter respectively. The constructs contained either the porcine signal peptide (pLHR-P10-297) or the insect signal peptide of melittin (pLHR-mel-319). Infected cells produced 1 x 10(5)-3 x 10(5) receptors/cell 3 days after infection. The recombinant LH receptor ectodomains produced were secreted in a biologically active form and bound the hormone with high affinity. Infected caterpillars produced a larger amount of active pLHR ectodomain that insect cells. The products were not secreted into the haemolymph however. Promoter and/or signal peptide modifications therefore enabled pLHR recombinant ectodomain secretion in a biologically active form, using the baculovirus-lepidopteran cell system. Moreover, moderate levels of expression seem to allow the production of biologically active ectodomain.

Published
1995
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