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

Abstract

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

Enhancement of Human Prolactin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

“…This confirmed the presence of about twice as much hPRL in the medium treated with 1 mM NaBu and the efficient adsorption of both the 25 kDa glycosylated (G-hPRL) and the 23 kDa nonglycosylated hPRL (NG-hPRL) isoforms by the cationic exchanger [31]. …”

Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis

“…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).…”

Современные Подходы К Диагностике И Лечению Гиперпролактинемии

“…Последовательность аминокислот схожа с таковой у плацентарного лактогена и гормона роста, что на протяжении длительно-го периода времени создавало трудности при идентификации пролактина. Период его полу-распада в крови составляет 20-30 мин [4][5][6].…”