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Purification of human recombinant anti-mullerian hormone and its derivatives
Alexandra Ya Rak1,2, Alexander V Trofimov1, Natalia V Pigareva1
1State Research Institute of Highly Pure Biopreparations, St Petersburg, Russian Federation.
Abstract:
Anti-mullerian hormone (AMH) is a cytokine of transforming growth factor β (TGF-β) superfamily able to induce apoptosis in cells bearing specific AMH type II receptors (AMHRII). AMHRII is overexpressed in some malignant cells, so at present recombinant AMH (rAMH) is considered as a new candidate antineoplastic drug. The use of rAMH may be especially effective in case of such severe diseases as ovarian, prostate and breast cancer. However, the development of a new drug is hampered by the laboriousness of obtaining highly purified rAMH and by the lack of data about the pharmacological characteristics of rAMH derivatives. In this work, we obtained preparations of prohormone, half-cleaved rAMH and a C-terminal fragment of rAMH, which was confirmed by qualitative and quantitative analyses. To obtain rAMH and its derivatives we used a previously developed highly effective producer strain containing the optimized human AMH gene. The production process has been divided into several stages: (a) rAMH biosynthesis in the bioreactor; (b) culture media preparation; (c) purification of rAMH and its derivatives using immunoaffinity chromatography and reversed-phase HPLC; (d) identification of the purified proteins by immunoblotting and analytical reversed-phase HPLC; and (e) evaluation of the hormone forms activity. The obtained proteins may be used in preclinical trials and in vitro study of rAMH derivatives properties.
Insights
Recombinant anti-Mullerian hormone (rAMH) and its derivatives were successfully produced and purified. These rAMH forms show potential as novel anti-cancer drugs for ovarian, prostate, and breast cancers.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Anti-Mullerian Hormone (AMH) is a TGF-β superfamily cytokine inducing apoptosis in cells with AMH type II receptors (AMHRII).
- AMHRII overexpression in certain cancers positions recombinant AMH (rAMH) as a potential antineoplastic agent.
- Challenges in rAMH drug development include laborious purification and limited data on derivative pharmacology.
Purpose of the Study:
- To produce and characterize prohormone, half-cleaved, and C-terminal rAMH fragments.
- To establish a robust method for obtaining highly purified rAMH derivatives.
- To provide materials for preclinical trials and in vitro studies of rAMH derivatives.
Main Methods:
- Utilized an optimized human AMH gene in a high-yield producer strain for biosynthesis.
- Employed bioreactor cultivation and subsequent purification via immunoaffinity chromatography and reversed-phase HPLC.
- Identified purified proteins using immunoblotting and analytical reversed-phase HPLC; evaluated hormone form activity.
Main Results:
- Successfully obtained preparations of prohormone, half-cleaved rAMH, and a C-terminal fragment.
- Confirmed protein identity and purity through qualitative and quantitative analyses.
- Developed a multi-stage process for efficient rAMH derivative production.
Conclusions:
- The developed methods enable the production of purified rAMH and its derivatives.
- These purified rAMH forms are suitable for preclinical investigations and in vitro pharmacological studies.
- This work facilitates the advancement of rAMH as a potential anti-cancer therapeutic.
