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United States Department of Agriculture

Agricultural Research Service


item Sherman, Gary
item Heilman, David
item Stocco, Douglas
item Ford, Johny
item Bohmfalk, John

Submitted to: Society for the Study of Reproduction Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 3/30/2001
Publication Date: 7/1/2001
Citation: Sherman, G., Heilman, D., Stocco, D., Ford, J.J., Bohmfalk, J. 2001. Production and partial purification of recombinant white rhinoceros luteinizing hormone [abstract]. Society for the Study of Reproduction Annual Meeting. 64 (Supplement 1):254.

Interpretive Summary:

Technical Abstract: In the present study we characterized an expression-enhanced cell line produced by methotrexate (MTX) -induced transgene amplification, and demonstrated immunoaffinity purification of rhinoceros luteinizing hormone (rec-wrLH). The expression system was comprised of the dihydrofolate reductase (DHFR) deficient host cell (DXB-11 CHO) and a dual expression vector (pDDEV) designed to constitutively express both alpha and beta subunit transgenes, as well as a functional DHFR minigene that served as a selectable marker. In addition, the alpha transgene cloning cassette was designed to fuse the FLAG immunoaffinity purification domain to the amino terminus of the mature wr-alpha subunit. Following initial transformation, 20 stably transfected cell lines testing positive for LH immunoactivity were selected for MTX-mediated amplification of the linked alpha/beta/DHFR transgene complex. Conditioned culture medium with the greatest biological activity was further characterized by heterologous radioimmnoassay (RIA) and western blot assay. Estimates of the concentration of rec-wrLH ranged from 1.5 ug/ml to10 ug/ml. Rapid isolation of heterodimeric rec-wrLH from conditioned media was achieved using an anti-FLAG monoclonal antibody (M2) resin and non-denaturing elution. Present studies indicate that recombinant expression systems can be used to produce quantities of rec-gonadotropins that are sufficient for assay development and pharmacological purposes.

Last Modified: 08/15/2017
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