Abstract
Prolactin (PRL) is a hormone produced by the pituitary gland with innumerable functions, such as lactation, reproduction, osmotic and immune regulation. The present work describes the synthesis of hPRL in human embryonic kidney (HEK293) cells, transiently transfected with the pcDNA-3.4-TOPO® vector carrying the hPRL cDNA. A concentration of ~ 20 mg/L, including glycosylated (G-hPRL) and non-glycosylated (NG-hPRL) human prolactin, was obtained, with ~ 19% of G-hPRL, which is higher than that observed in CHO-derived hPRL (~ 10%) and falling within the wide range of 5–30% reported for pituitary-derived hPRL. N-Glycoprofiling analysis of G-hPRL provided: (i) identification of each N-glycan structure and relative intensity; (ii) average N-glycan mass; (iii) molecular mass of the whole glycoprotein and relative carbohydrate mass fraction; (iv) mass fraction of each monosaccharide. The data obtained were compared to pituitary- and CHO-derived G-hPRL. The whole MM of HEK-derived G-hPRL, determined via MALDI–TOF-MS, was 25,123 Da, which is 0.88% higher than pit- and 0.61% higher than CHO-derived G-hPRL. The main difference with the latter was due to sialylation, which was ~ sevenfold lower, but slightly higher than that observed in native G-hPRL. The “in vitro” bioactivity of HEK-G-hPRL was ~ fourfold lower than that of native G-hPRL, with which it had in common also the number of N-glycan structures.
Highlights
Human prolactin is a pituitary hormone, which has an essential role on mammopoiesis
Considering expression efficiency and the higher yields obtained, our studies were carried out just utilizing pcDNA 3.4 TOPO-Human prolactin (hPRL) vector in transiently transfected HEK293F suspension cells
Human recombinant prolactin has been synthesized for the first time in a human host: HEK293 cells, either adherent (A) or in suspension (S) culture, using three different expression vectors
Summary
Human prolactin (hPRL) is a pituitary hormone, which has an essential role on mammopoiesis. Especially for diagnostic purposes, is related to lactation problems and infertility in women and to the fact that elevated circulating or locally produced hPRL levels are associated with the risk of breast and hPRL is a 199 amino acid-polypeptide, whose theoretical molecular mass (MM) of 22,897.75 Da, calculated from the amino acid sequence (Wu et al 2003), has been perfectly confirmed by our research group via MALDI–TOF-MS on different CHO-, C127-, E. coli- and pituitary-derived preparations This provided an average value of 22,910.3 Da, which is only 0.055% higher than the theoretical one (Capone et al 2015; Heller et al 2010; Soares et al 2006; Wu et al 2003). We are dealing with one of the simplest types of
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