Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations.
Analytical techniques
Chromatographic purification
Glycosylation analysis
Recombinant equine chorionic gonadotropin
Journal
The protein journal
ISSN: 1875-8355
Titre abrégé: Protein J
Pays: Netherlands
ID NLM: 101212092
Informations de publication
Date de publication:
02 2023
02 2023
Historique:
accepted:
11
01
2023
pubmed:
19
1
2023
medline:
3
3
2023
entrez:
18
1
2023
Statut:
ppublish
Résumé
Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purified PMSG. To gain insight into the structural and functional characteristics of reCG, a bioinformatics analysis was performed. An exhaustive characterization comprising the determination of the purity degree, aggregates and nicked forms through SDS-PAGE, RP-HPLC and SEC-HPLC was performed. Higher order structures were studied by fluorescence spectroscopy and SEC-HPLC. Isoforms profile were analyzed by isoelectric focusing. Glycosylation analysis was performed through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC. Slight differences between the purified recombinant hormones were found. However, recombinant molecules and PMSG exhibited variations in the glycosylation pattern. In fact, differences in sialic acid content between two commercial preparations of PMSG were also obtained, which could lead to differences in their biological potency. These results show the importance of having a standardized production process, as occurs in a recombinant protein bioprocess. Besides, our results reflect the importance of the glycan moieties on eCG conformation and hence in its biological activity, preventing denaturing processes such as aggregation.
Identifiants
pubmed: 36652139
doi: 10.1007/s10930-023-10092-x
pii: 10.1007/s10930-023-10092-x
doi:
Substances chimiques
Gonadotropins, Equine
0
Chorionic Gonadotropin
0
Recombinant Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
24-36Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
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