Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Gerard Castro-Linares, Jeffrey Den Haan, Francois Iv, Carla Silva Martins, Aurélie Bertin*, Manos Mavrakis, Gijsje H. Koenderink

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

Original languageEnglish
Article numbere63871
Number of pages20
JournalJournal of Visualized Experiments
Volume2022
Issue number184
DOIs
Publication statusPublished - 2022

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