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BMC Biotechnol. 2013 Jul 26;13:60. doi: 10.1186/1472-6750-13-60.

An efficient protocol for the purification and labeling of entire yeast septin rods from E.coli for quantitative in vitro experimentation.

Author information

1
Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, D-89081 Ulm, Germany.

Abstract

BACKGROUND:

The detailed understanding of the functions and mechanisms of the actin and microtubuli cytoskeleton depended, besides innovative methods in live cell imaging, on the purification and labeling of its constituents. This allowed researchers to quantitatively measure filament stability, the rates of filament turnover as well as the determination of the influence of cofactors on filament formation and structure. Septins form the least understood class of cytoskeletal structures in nearly all eukaryotic cells so far examined. In yeast, they comprise a family of proteins (Cdc3, Cdc10, Cdc11, Cdc12, Shs1) that form a co-polymeric, ring-like structure beneath the membrane. This ring serves as a template for the formation of a new bud neck and as a landing pat for proteins involved in polar growth and cytokinesis. Further progress in investigating the mechanisms of septin-structure formation and regulation is hampered by the lack of protocols to modify homogenous samples of purified septins with useful probes for in vitro biochemical studies.

RESULTS:

We present a protocol for the purification and labeling of yeast septin rods. The four individual septin subunits were co-expressed in E.coli. One subunit of the septin polymer was expressed as SNAP tag fusion protein allowing for rapid and stoichiometric labeling with derivatized Benzylguanine (BG). To demonstrate the applicability of our approach, we introduced two different SNAP tag substrates: septin rods labeled with fluorescent BG compounds enabled us to monitor the formation of filaments by fluorescence microscopy whereas BG-biotin was used to couple septin rods to a sensor chip for quantitative surface plasmon resonance binding experiments. In a first application, we determined the affinity and the binding kinetics of the yeast protein Bni5 to the individually coupled septin rods. In a further application we could demonstrate that a once formed septin rod hardly exchange its subunits.

CONCLUSIONS:

The herein introduced protocol of purifying SNAP tag modified septins from E.coli allowed us to derivatize the obtained septin rods with probes for the further in vitro characterization of this class of cytoskeletal elements. The availability of a very diverse set of SNAP tag substrates should open the way to investigate different aspects of septin biochemistry in mechanistic detail.

PMID:
23889817
PMCID:
PMC3765318
DOI:
10.1186/1472-6750-13-60
[Indexed for MEDLINE]
Free PMC Article

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