High-level synthesis of human prolactin in Chinese-hamster ovary cells

Two eukaryotic human prolactin (hPRL) expression vectors, based on a select able dihydrofolate reductase (dhfr) marker, were used to transfect dhfr Chi nese-hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatit is-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation, The other, pEDdc-hPRL, carries the encephalomyocard itis virus leader. sequence coupled to hPRL cDNA to provide high-level prot ein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA, Without methotrexate (MTX) amplification, p658-hPRL-t ransfected stable cell lines, secreting up to approximate to 10 mu g of hPR L/10(6) cells per day, could be rapidly obtained; production By pEDdc-hPRL- transfected cells was about 10-fold lower. However, a three-step MTX amplif ication of the latter led to clones secreting up to approximate to 30 mu g of hPRL/10(6) cells per day. A pilot production using a hollow-fibre biorea ctor indicated that highly concentrated hormone levels in the medium could be databased, with a production of up to 150 mu g of hPRL/ml per day. SDS/P AGE analysis indicated that recombinant hPRL contained approximate to 10% g lycosylated PRL, Chromatographically purified non-glycosylated and glycosyl ated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay), This appears to be the first report describing product ion and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.