N-glycoprofiling analysis in a simple glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin.

Human prolactin (hPRL) is a polypeptide hormone occurring in the non-glycosylated (NG-hPRL) and glycosylated (G-hPRL) forms, with MM of approximately 23 and 25kDa, respectively. It has a single, partially occupied N-glycosylation site located at Asn-31, which makes it a particularly simple and interesting model for glycosylation studies. The bioactivity of G-hPRL is lower than that of NG-hPRL (by ca. 4-fold) and its physiological function is not clear. However, carbohydrate moieties generally play important roles in the biosynthesis, secretion, biological activity, and plasma survival of glycohormones and can vary depending on the host cell. The main objective of this study was to determine the N-glycan structures present in native, pituitary G-hPRL and compare them with those present in the recombinant hormone. To obtain recombinant G-hPRL, genetically modified Chinese hamster ovary cells (CHO), adapted to growth in suspension, were treated with cycloheximide, thus increasing the glycosylation site occupancy from 5.5% to 38.3%, thereby facilitating G-hPRL purification. CHO cell-derived G-hPRL (CHO-G-hPRL) was compared to pituitary G-hPRL (pit-G-hPRL) especially with regard to N-glycoprofiling. Among the main differences found in the pituitary sample were an extremely low presence of sialylated (1.7%) and a high percentage of sulfated (74.0%) and of fucosylated (90.5%) glycans. A ∼6-fold lower in vitro bioactivity and a higher clearance rate in mice were also found for pit-G-hPRL versus CHO-G-hPRL. N-Glycan profiling proved to be a useful and accurate methodology also for MM and carbohydrate content determination for the two G-hPRL preparations, in good agreement with the values obtained directly via MALDI-TOF-MS.

Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space.

Prolactin (PRL) is a pleiotropic hormone produced by lactotroph cells of the anterior pituitary gland and is mainly related to lactation control and reproduction. Recombinant mouse prolactin (r-mPRL), never obtained in its authentic form, can be very useful for research and tests in animal models, in which human prolactin (hPRL) is usually employed in a heterologous mode. Synthesis of r-mPRL was carried out here via secretion in Escherichia coli periplasmic space using a plasmid containing mPRL cDNA joined to the DsbA signal peptide sequence under the control of a constitutive major leftward promoter of the bacteriophage λ (λPL). Fermentation in a pilot bioreactor was carried out at 30°C, with 6 H of induction at 37°C, reaching an optical density of 23 A600 units, a specific yield of 0.06-0.1 µg mPRL/(mL A600), and a concentration of up to 2.2 µg/mL. Even with such a low yield and a poor mass fraction, r-mPRL was purified via a three-step laboratory process based on hydrophobic chromatography, reversed-phase high-performance liquid chromatography, and high-performance size-exclusion chromatography (HPSEC). The purified hormone was then characterized using SDS-PAGE, Western blotting, and HPSEC and showed, by Nb2 rat lymphoma cell proliferation assay, a bioactivity of 39.5 IU/mg, determined against the International Standard of recombinant hPRL [World Health Organization (WHO)-97/714].