Recombinant expression and secretion of a natural splicing variant containing the ectodomain of porcine LH receptor in HC11 mammary epithelial cells.
Large-scale synthesis of active recombinant porcine luteinizing hormone/chorionic gonadotropin receptor (pLHR) is required for biophysical and structural studies. This study was undertaken to improve expression of the corresponding cDNA already obtained with a number of other systems, (i) by turning to cells from mammalian origin able to perform adequate glycosylation, (ii) by using an expression vector containing the acknowledged high-performance rabbit WAP gene upstream region together with transcription and translation stimulating sequences, and (iii) by expressing natural splicing variants. Selection of the transfected HC11 cells was performed in terms of pLHR expression using specific radioligand binding and immunoradiometric assays. Secretion of pLHR ectodomain into the culture medium of the HC11 clones was quantified, and reached 70 ng/ml, which represents the highest active amount ever produced. However, this level of expression was relatively low in comparison to that currently observed with bGH cDNA used as reporter gene. Additional investigations were performed in order to gain further insight into the limitation of the production of pLHR relative to bovine or human growth hormone using the same expression system. A high number of copies of cDNA in the genome of HC11 cells was found, provided that an antibiotic selection pressure was maintained to avoid drifting. The low mRNA levels detected for pLHR relative to hGH mRNAs correlate well with the relative protein production levels. They could arise from poor stability of mRNAs, a fact already observed for the natural receptor in gonadal cells. These results thus constitute a promising indicator for possible expression of pLHR in the milk of transgenic animals.
Pajot-Augy
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E
Unité Récepteurs et Communication Cellulaire, INRA, Laboratoire de Biologie Cellulaire et Moléculaire, Jouy-en-Josas, France. pajot@biotec.jouy.inra.fr
Attal
J
J
Théron
M C
MC
Devinoy
E
E
Fontaine
M L
ML
Houdebine
L M
LM
Salesse
R
R
D000818:Animals; D002417:Cattle; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006031:Glycosylation; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D015592:Immunoradiometric Assay; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D015336:Molecular Probe Techniques; D011388:Prolactin / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D011869:Radioligand Assay; D011974:Receptors, LH / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013045:Species Specificity; D013552:Swine / Q000235:genetics*
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D012333:RNA, Messenger; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D003907:Dexamethasone; D011388:Prolactin; D013006:Growth Hormone