Abstract
Background Prolactin (PRL) is a peptide hormone related to innumerous physiological functions such as lactation, reproduction, osmotic and immune regulation [1]. In order to evaluate new drugs many tests are conducted in mouse models. Use of prolactin from different origin in mouse model can result in differences in biological activities [2]. Only 61% sequence homology is observed between mouse PRL (197 amino acid residues, 22.5 kDa) and human PRL (199 amino acid residues, 23 kDa). Authentic mouse PRL, without initial methionine, expressed in periplasmic fluid of E. coli has already been characterized and, as known, it does not present the glycosylated isoform (G-PRL) [3]. About 5-30% of PRL from mammalian pituitaries is glycosylated [1]. Physiologically, nonglycosylated and glycosylated PRL presented differences concerning lactogenic activity, binding affinity and mitogenicity. The objective of this work was to produce mPRL in CHO dhfr-cells, with gene amplification based on methotrexate addition, so to obtain the glycosylated and non-glycosylated forms of mPRL directly secreted into serum free media.
Highlights
Prolactin (PRL) is a peptide hormone related to innumerous physiological functions such as lactation, reproduction, osmotic and immune regulation [1]
After DNA sequencing and analysis in 1% agarose of the fragments obtained by digestion with the restriction enzymes XbaI, EcoRI, BamHI, and XhoI confirming the correct DNA sequence, Lipofectamine was used to introduce the plasmid pEDdc-mPRL into CHO dhfr- cells
Construction of pEDdc-mPRL plasmid was confirmed by DNA sequencing and restriction analysis
Summary
Prolactin (PRL) is a peptide hormone related to innumerous physiological functions such as lactation, reproduction, osmotic and immune regulation [1]. In order to evaluate new drugs many tests are conducted in mouse models. Use of prolactin from different origin in mouse model can result in differences in biological activities [2]. 61% sequence homology is observed between mouse PRL (197 amino acid residues, 22.5 kDa) and human PRL (199 amino acid residues, 23 kDa). Authentic mouse PRL, without initial methionine, expressed in periplasmic fluid of E. coli has already been characterized and, as known, it does not present the glycosylated isoform (G-PRL) [3]. About 5-30% of PRL from mammalian pituitaries is glycosylated [1]. Nonglycosylated and glycosylated PRL presented differences concerning lactogenic activity, binding affinity and mitogenicity. The objective of this work was to produce mPRL in CHO dhfr-cells, with gene amplification based on methotrexate addition, so to obtain the glycosylated and non-glycosylated forms of mPRL directly secreted into serum free media
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