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 hormon e/chorionic gonadotropin receptor (pLHR) is required for biophysical a nd structural studies. This study was undertaken to improve expression of the corresponding cDNA already obtained with a number of other sys tems, (i) by turning to cells from mammalian origin able to perform ad equate glycosylation, (ii) by using an expression vector containing th e acknowledged high-performance rabbit WAP gene upstream region togeth er 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 r adioligand binding and immunoradiometric assays. Secretion of pLHR ect odomain into the culture medium of the HC11 clones was quantified, and reached 70 ng/ml, which represents the highest active amount ever pro duced. However, this level of expression was relatively low in compari son to that currently observed with bGH cDNA used as reporter gene. Ad ditional investigations were performed in order to gain further insigh t into the limitation of the production of pLHR relative to bovine or human growth hormone using the same expression system. A high number o f 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 wel l with the relative protein production levels. They could arise from p oor stability of mRNAs, a fact already observed for the natural recept or in gonadal cells. These results thus constitute a promising indicat or for possible expression of pLHR in the milk of transgenic animals. (C) 1997 Academic Press.