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.
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.
Bozon
V
V
Unité d'Ingénierie des Protéines, INRA-Biotechnologies, Jouy-en-Josas, France.
Remy
J J
JJ
Pajot-Augy
E
E
Couture
L
L
Biache
G
G
Severini
M
M
Salesse
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors; D007700:Kinetics; D007814:Larva; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008555:Melitten / Q000235:genetics*; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D013552:Swine
D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D008555:Melitten; D007986:Luteinizing Hormone