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The protein journal2023; 42(1); 24-36; doi: 10.1007/s10930-023-10092-x

Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations.

Abstract: 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.
© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
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Publication Date: 2023-01-18 PubMed ID: 36652139PubMed Central: 5336677DOI: 10.1007/s10930-023-10092-xGoogle Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study is about the physicochemical characterization of a recombinant equine chorionic gonadotropin (eCG), compared to commercial preparations of pregnant mare serum gonadotropin (PMSG). The research highlights the differences between the two, especially in terms of their glycosylation patterns, and emphasizes the importance of standardizing production processes.

Research Background

  • This research is centered around the hormone eCG, widely used in artificial ovulation and superovulation procedures to boost reproductive performance in livestock.
  • Until recently, the available eCG-based products for veterinary use had been partially purified versions of PMSG.
  • The study therefore aims to understand the structural and functional attributes of a bioactive recombinant eCG (reCG) that was produced in suspension CHO-K1 cells.

Methods Used

  • The recombinant eCG was purified using different chromatographic methods, including hydrophobic interaction chromatography and reverse-phase (RP)-HPLC, and then compared with a RP-HPLC-purified PMSG.
  • The researchers also executed a bioinformatics analysis to get a deeper understanding of reCG.
  • Several tests were carried out to determine the purity degree, aggregates, nicked forms, and higher order structures. Methods used included SDS-PAGE, RP-HPLC, and SEC-HPLC.
  • Glycosylation analysis was done through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC.

Research Findings

  • There were some differences found between the purified recombinant hormones. However, variations in the glycosylation pattern were observed between the recombinant molecules and PMSG.
  • Differences in sialic acid content were discovered between two commercial PMSG preparations, which could potentially lead to discrepancies in their biological potency.

Significance of the Study

  • The findings highlight the importance of standardizing production processes, especially in the bioprocess of recombinant proteins.
  • The results also underscore the significance of the glycan moieties on the structure of eCG and its biological activity, thereby preventing denaturing processes such as aggregation.

Cite This Article

APA
Rodríguez MC, Mussio PE, Villarraza J, Tardivo MB, Antuña S, Fontana D, Ceaglio N, Prieto C. (2023). Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations. Protein J, 42(1), 24-36. https://doi.org/10.1007/s10930-023-10092-x

Publication

The protein journal
ISSN: 1875-8355
NlmUniqueID: 101212092
Country: Netherlands
Language: English
Volume: 42
Issue: 1
Pages: 24-36

Researcher Affiliations

Rodríguez, María Celeste
  • UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina. mcrodriguez@fbcb.unl.edu.ar.
Mussio, Pablo Esteban
  • UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina.
Villarraza, Javier
  • UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina.
Tardivo, María Belén
  • Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina.
Antuña, Sebastián
  • Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina.
Fontana, Diego
  • UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina.
Ceaglio, Natalia
  • UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Cell Culture Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina.
Prieto, Claudio
  • UNL, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Biotechnological Development Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina.
  • Biotecnofe S.A. PTLC, Ruta 168 Pje El Pozo, (3000), Santa Fe, Argentina.
  • Cellargen Biotech SRL, FBCB (School of Biochemistry and Biological Sciences), Biotechnological Development Laboratory, Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA), Santa Fe, Argentina.

MeSH Terms

  • Pregnancy
  • Female
  • Animals
  • Horses
  • Gonadotropins, Equine
  • Chorionic Gonadotropin
  • Glycosylation
  • Recombinant Proteins / chemistry
  • Electrophoresis, Polyacrylamide Gel

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