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bioRxiv · 10.64898/2026.09.10.750725

An efficient system for production of highly purified inhibins using mammalian cell expression and tandem affinity purification

Buddle, E. R. S.·Hinck, C. S.·Lin, Y.-F.·Wang, Y.·Krzysiak, T. C.·Bernard, D. J.·Hinck, A. P.
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Abstract

Inhibin A and B (InhA and InhB) are potent negative regulators of follicle-stimulating hormone (FSH), which is essential for female fertility. InhA and InhB are heterodimeric transforming growth factor {beta} (TGF-{beta}) family ligands consisting of an inhibin (inh) subunit disulfide linked to an inhibin {beta}A or {beta}B (inh{beta}A, inh{beta}B) subunit, respectively. During purification, inhibins must be separated from activins (inh{beta}A or inh{beta}B homodimers), which always form when producing inhibins, and have the opposite effect on FSH synthesis. Current inhibin purification methods are technically challenging, require reagents that are not generally available, result in low purity or contamination with activins, or employ substitutions within the growth factor domain to favor inhibin formation over activin. We developed a simple and high-yielding expression and purification system for inhibins by transfection of suspension-cultured mammalian cells and tandem affinity chromatography, followed by reverse-phase purification of the mature ligands. We purified 1.4 mg (n = 3, SD = 0.47) and 0.093 mg (n = 3, SD = 0.043) of wild-type InhA and InhB per liter of culture medium with little to no contaminating activin A or B, at equal to higher purity and bioactivity than commercially available inhibins. Thus, we present a simple, robust, and versatile system for producing inhibins to interrogate their functions and mechanisms of action in vitro and in vivo.

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Buddle, E. R. S., Hinck, C. S., Lin, Y.-F., Wang, Y., Krzysiak, T. C., Bernard, D. J., Hinck, A. P.. 2026-09-14. An efficient system for production of highly purified inhibins using mammalian cell expression and tandem affinity purification. https://doi.org/10.64898/2026.09.10.750725

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