Dataset Information

Isolation and thermal stabilization of mouse ferroportin.

ABSTRACT: Ferroportin (Fpn) is an essential mammalian iron transporter that is negatively regulated by the hormone hepcidin. Our current molecular understanding of Fpn-mediated iron efflux and regulation is limited due to a lack of biochemical, biophysical and high-resolution structural studies. A critical step towards understanding the transport mechanism of Fpn is to obtain sufficient quantities of pure and stable protein for downstream studies. As such, we detail here an expression and purification protocol for mouse Fpn yielding milligram quantities of pure protein. We have generated deletion constructs exhibiting enhanced thermal stability and which retained iron-transport activity and hepcidin responsiveness, providing a platform for further biophysical studies of Fpn.

SUBMITTER: Deshpande CN

PROVIDER: S-EPMC7780100 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Isolation and thermal stabilization of mouse ferroportin.

Deshpande Chandrika N CN Azucenas Corbin R CR Qiao Bo B Nomura Norimichi N Xin Vicky V Font Josep J Iwata So S Ganz Tomas T Nemeth Elizabeta E Mackenzie Bryan B Jormakka Mika M

FEBS open bio 20201217 1

Ferroportin (Fpn) is an essential mammalian iron transporter that is negatively regulated by the hormone hepcidin. Our current molecular understanding of Fpn-mediated iron efflux and regulation is limited due to a lack of biochemical, biophysical and high-resolution structural studies. A critical step towards understanding the transport mechanism of Fpn is to obtain sufficient quantities of pure and stable protein for downstream studies. As such, we detail here an expression and purification pro ...[more]

PMID: 33190422

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Project description:Enzymatic degradation is a major concern in peptide analysis. Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations. It is therefore vital to find reliable, reproducible, and easy-to-use procedures to inhibit enzymatic activity in fresh tissues before subjecting them to qualitative and quantitative analyses. The aim of this study was to test a benchtop thermal stabilization method to optimize measurement of endogenous opioids in brain tissue. Endogenous opioid peptides are generated from precursor proteins through multiple enzymatic steps that include conversion of one bioactive peptide to another, often with a different function. Ex vivo metabolism may, therefore, lead to erroneous functional interpretations. The efficacy of heat stabilization was systematically evaluated in a number of postmortem handling procedures. Dynorphin B (DYNB), Leu-enkephalin-Arg(6) (LARG), and Met-enkephalin-Arg(6)-Phe(7) (MEAP) were measured by radioimmunoassay in rat hypothalamus, striatum (STR), and cingulate cortex (CCX). Also, simplified extraction protocols for stabilized tissue were tested. Stabilization affected all peptide levels to varying degrees compared to those prepared by standard dissection and tissue handling procedures. Stabilization increased DYNB in hypothalamus, but not STR or CCX, whereas LARG generally decreased. MEAP increased in hypothalamus after all stabilization procedures, whereas for STR and CCX, the effect was dependent on the time point for stabilization. The efficacy of stabilization allowed samples to be left for 2 hours in room temperature (20°C) without changes in peptide levels. This study shows that conductive heat transfer is an easy-to-use and efficient procedure for the preservation of the molecular composition in biological samples. Region- and peptide-specific critical steps were identified and stabilization enabled the optimization of tissue handling and opioid peptide analysis. The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

Dataset Information

Isolation and thermal stabilization of mouse ferroportin.

Publications

Isolation and thermal stabilization of mouse ferroportin.

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