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  Purification and characterization of eukaryotic ATP-dependent transporters homologously expressed in Pichia pastoris for structural studies by cryo-electron microscopy

Kalavacherla, T., Buschmann, S., Schleker, E. S. M., Michel, H., & Reinhart, C. (2023). Purification and characterization of eukaryotic ATP-dependent transporters homologously expressed in Pichia pastoris for structural studies by cryo-electron microscopy. Protein Expression and Purification, 204: 106230. doi:10.1016/j.pep.2023.106230.

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 Creators:
Kalavacherla, Tejaswi1, Author                 
Buschmann, Sabine1, Author                 
Schleker, E. Sabine M.1, Author                 
Michel, Hartmut1, Author                 
Reinhart, Christoph1, Author                 
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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 Abstract: Membrane proteins play an essential role in all living organisms. Although there have been numerous efforts in the past to elucidate the structure and function of eukaryotic primary active transporters, knowledge about the majority of these membrane proteins is still minimal. This is often due to their low availability and complex handling. In this study, we homologously expressed three ATP-dependent transport proteins, STE6-2p, NEO1-p, and YPK9-p, in Pichia pastoris and subsequently optimized the solubilization and purification processes. Sequential use of different mild detergents and utilization of hydrophilic matrices in the purification procedure allowed us to obtain all three transporters monodisperse and in high purity, enabling initial structural analysis by cryo-electron microscopy. Using the respective substrates, we determined the specific activity of all target proteins using an ATPase assay. This study opens the door to further functional and structural studies of this pharmacologically important class of membrane proteins.

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Language(s): eng - English
 Dates: 2022-12-222022-10-212023-01-012023-01-082023-04
 Publication Status: Issued
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.pep.2023.106230
PMID: 36632890
 Degree: -

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Title: Protein Expression and Purification
Source Genre: Journal
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Publ. Info: San Diego : Academic Press
Pages: - Volume / Issue: 204 Sequence Number: 106230 Start / End Page: - Identifier: ISSN: 1046-5928
CoNE: https://pure.mpg.de/cone/journals/resource/954922650158