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Biotechnology and Applied Biochemistry (2000) 32, (127–135) (Printed in Great Britain)

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

Carlos R. J. Soares*, Ligia Morganti*, Brigitte Miloux†, Jan H. Lupker†, Pascual Ferrara† and Paolo Bartolini*¹

*Biotechnology Department, National Nuclear Energy Commission (IPEN-CNEN), Travessa R-400, Cidade Universitária 05508-900, São Paulo, Brazil, and †Sanofi Recherche, Labège-Innopole, Voie no. 1, B.P. 137, 31676, France

Abbreviations used: hPRL, human prolactin; rec-hPRL, recombinant hPRL; G-hPRL, glycosylated hPRL; NG-hPRL, non-glycosylated hPRL; CHO, Chinese-hamster ovary; dhfr, dihydrofolate reductase; MTX, methotrexate; MEM, minimal essential medium; FBS, fetal bovine serum; SP, sulphopropyl; CV, coefficient of variation.

¹ To whom correspondence should be addressed (e-mail pbartoli@net.ipen.br).

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 10 µg of hPRL/10⁶ 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 30 µg of hPRL/10⁶ 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 µg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained 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.

Received 15 June 2000; accepted 28 June 2000

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