ABSTRACT
Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.
Subject(s)
Anti-Mullerian Hormone , Chromatography, High Pressure Liquid/methods , Recombinant Proteins , Animals , Anti-Mullerian Hormone/chemistry , Anti-Mullerian Hormone/isolation & purification , Anti-Mullerian Hormone/pharmacology , CHO Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatography, Reverse-Phase/methods , Cricetinae , Cricetulus , Humans , Hydrophobic and Hydrophilic Interactions , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacologyABSTRACT
Anti-mullerian hormone (AMH) is a cytokine of transforming growth factor ß (TGF-ß) 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.
