Purification strategies for equine Chorionic Gonadotropin
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Date
2022
Authors
Boneschans, Martie
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
Equine Chorionic Gonadotropin (eCG) is a glycoprotein hormone secreted by the endometrial cups of pregnant mares during days 37 to 120 of gestation. In equids it exhibits luteinizing hormone (LH) activity, while in non-equine species eCG exhibits both LH - and follicle stimulating hormone (FSH) like activity. Due to the high sialic acid content of eCG the 60 kD protein possess a long half-life in mammalian plasma. This, together with its dual hormonal activity (LH and FSH) makes eCG an extremely effective hormone for use in animal reproduction. Recombinant production of the hormone is still in its initial phases, therefore eCG, for commercial use, is still primarily purified from the serum of pregnant mares. Equine serum albumin remains the biggest challenge in the purification of eCG due to its relative abundance in the serum and similar molecular weight (70 kDa). This study investigated some of the strategies involved in the isolation of eCG in order to identify possible areas for improvement.
We compared three different acid precipitation steps as an initial phase in the isolation of eCG from eCG-spiked equine serum. Precipitation with metaphosphoric acid (MPA) showed the highest purification factor compared to precipitation with 15% (v/v) –and 25% (v/v) trichloroacetic acid (TCA) but resulted in high eCG losses. We also investigated the use of albumin affinity chromatography in the purification process but found that both albumin and eCG bound to the albumin affinity resin, raising our suspicions about possible interaction between the proteins. Ultrafiltration, using filtration units with a MWCO of 30 kDa, proved an ineffective method in the isolation of eCG as we found that eCG exhibits an affinity for the porous membrane of the filtration unit. During cation exchange chromatography we found that when eCG was mixed with BSA, the proteins exhibited changes in their column binding properties. This was confirmed with anion exchange where neither a continuous salt – nor pH gradient could separate eCG and albumin. This further supports our notion of a possible interaction between the two proteins as albumin is widely known as a carrier protein for thyroid hormones and other molecules. We found that Lectin affinity (LAC) - and hydrophobic interaction chromatography (HIC) did succeed in separating eCG and albumin but we could not elute most of the eCG from the LAC column, therefore future work should include further optimization of the method. HIC was performed on small scale and future work should look in to using higher concentrations of protein.
Description
Dissertation (M.Sc.(Biochemistry))--University of the Free State, 2022
Keywords
eCG, equine chorionic gonadotropin, glycoprotein hormone, purification, protein precipitation, affinity chromatography