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

Abstract

Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA. Without methotrexate (MTX) amplification, p658-hPRL-transfected stable cell lines, secreting up to approximately 10 microg of hPRL/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 amplification of the latter led to clones secreting up to approximately 30 microg of hPRL/10(6) cells per day. A pilot production using a hollow-fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 microg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained approximately 10% glycosylated PRL. Chromatographically purified non-glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay). This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

“…The clone expressing hPRL, derived from CHO dhfr -cells (DUKX-B11) that had been transfected with the vector pEDdc-hPRL, was obtained in our Laboratory (Soares et al, 2000). Cells were thawed into T-flasks (75 cm 2 , from Corning Costar Corporation, Cambridge, MA) containing ␣-MEM with l-glutamine and without ribonucleosides and deoxyribonucleosides, supplemented with 10% dialyzed fetal bovine serum (dFBS), 40 g ml −1 gentamycin, 4 g l −1 glucose and 100 nM methotrexate.…”

“…Unfortunately, the physiological significance of this isoform has not yet been well elucidated, even though it has been postulated that hPRL glycosylation might possibly modulate the bioactivity of the circulating pool of the hormone, perhaps by selectively down-regulating PRL action at individual target tissues (Sinha, 1995;Freeman et al, 2000;Goffin et al, 2002). For these reasons, we and others believe that this and other prolactin variants are needed in purified and characterized form in order to develop proper physiological and clinical studies directed towards the understanding of the more than 300 separate actions of this hormone, whose receptor distribution has been defined as being "quasi-ubiquitous" (Young et al, 1990;Price et al, 1995;Sinha, 1995;Freeman et al, 2000;Soares et al, 2000;Goffin et al, 2002). Impetus for the present investigation was provided by the work of Shelikoff et al (1994), who demonstrated that the addition of a protein synthesis inhibitor (cycloheximide) to C127 cell culture increased recombinant hPRL glycosylation site occupancy.…”

“…Glycosylated human prolactin (G-hPRL) was first isolated and purified from human pituitaries by Lewis et al (1985), with an estimated molecular mass of ∼25,000 Da and an immunological and biological activity of 25-50% that of non-glycosylated (NG)-hPRL (Pellegrini et al, 1988;Lewis et al, 1989;Sinha et al, 1991;Hoffmann et al, 1992;Price et al, 1995;Soares et al, 2000). The presence of a unique and partially occupied glycosylation site in Asn-31 in human, monkey, ovine, porcine, dromedary, equine and whale PRL (Sinha, 1995;Butnev et al, 1996) makes it an ideal model of glycosylation for N-glycan studies since it exhibits the simplest type of glycosylation macroheterogeneity, with an occupancy range of 10-30% of G-hPRL relative to the total hPRL of either pituitary or recombinant origin (Shelikoff et al, 1994(Shelikoff et al, , 1996Price et al, 1995;Sinha, 1995; Abbreviations: CHO, Chinese hamster ovary; CRS, chemical reference standard; hPRL, human prolactin; HPSEC, size-exclusion HPLC; MALDI-TOF-MS, matrixassisted laser desorption ionization time-of-flight mass spectrometry; Mr, relative molecular mass; PAGE, polyacrylamide-gel electrophoresis; SDS, sodium dodecyl sulfate; RP-HPLC, reversed-phase HPLC; tR, retention time.…”

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

“…The GCR cDNA was cloned into the pED dicistronic expression vector, which is commonly used to obtain high-expression levels of heterologous proteins in mammalian cells [45–47], and positive clones were isolated and amplified with increasing MTX concentration up to 700 nM MTX. Probably due to the inability of the transfected cells to further amplify the dhfr gene [28], higher concentration of MTX (1200 nM) caused cell death.…”

“…Although the highest productivity achieved for secreted GCR was calculated as 5.14 pg/cell/day, higher expression levels have been reported for other recombinant proteins in CHO cells [45, 47]. Here, it should be taken into account the fact that GCR is a membrane-associated glycoprotein secreted at low level or rather not secreted under natural conditions [37].…”