Product uses
- Identification and characterization of FtsZ binding proteins
- Characterization of FtsZ dynamics
- Developing FtsZ ligands that may be used as anti-bacterial agents.
Material
FtsZ is a bacterial cytoskeletal protein that is essential for cell division many prokaryotes (1). It has been shown to be a bacterial homolog of eukaryotic tubulin, based both on a low sequence identity and a striking structural similarity (2). Just like eukaryotic tubulin, FtsZ polymerizes as well as binds and hydrolyzes GTP in a polymerization dependent manner.
Cytoskeleton, Inc's FtsZ protein has been expressed in E. coli and purified by DEAE cellulose chromatography as described by Lu and coworkers (3). It is active in both polymerization and GTPase assays. FtsZ has an approximate molecular weight of 50 kDa.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 10% polyacrylamide gel. FtsZ protein was found to be >90% pure (see Figure 1). The major contaminants are E.coli proteins that co-purify with FtsZ. These contaminants may result in a slightly higher GTPase activity.
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Figure 1: A 100 µg sample of E.c. FtsZ protein was separated on a 4-20% gradient SDS-PAGE gel and stained with Coomassie blue. Lane 1, 100µg FTZ01. SeeBlue molecular weight markers are from Invitrogen.
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Biological activity
The biological activity of FtsZ is determined in two ways, first from its ability to efficiently polymerize into filaments in vitro in the presence of Ca2+ and GTP, and secondly to readily hydrolyze GTP to GDP and Pi. Polymerization by Ca2+:GTP results in bundles of FtsZ filaments that sediment at 50,000 x g. GTPase activity is determined by measuring the Pi released during a 5min reaction, by using the malachite green reagent (Cat. # BK050). In these reactions, more than 70% of our FtsZ protein polymerizes and it has a GTPase activity above 0.3 mol GTP/mol FtsZ/min.
References
- Bramhill, D. (1997) Bacterial cell division. Annu. Rev. Cell Dev. Biol. 13, 395-424.
- Erickson, H. P. (1998) Atomic structures of tubulin and FtsZ. Trends Cell Biol. 8, 133-137.
- Lu, C., Stricker, J., and Erickson, H. P. (2001) Site-specific mutations of FtsZ--effects on GTPase and in vitro assembly. BMC Microbiol. 1, 7.
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
Product Description
MSDS
Datasheet
Cat #
FtsZ protein: E. coli recombinant
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FTZ01
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Coming soon! For the most recent publications citing this product, please contact our Technical Service department at tservice@cytoskeleton.com
Question 1: Are other FtsZ proteins available besides the FtsZ from E. Coli?
Answer 1: Yes, Cytoskeleton offers custom protein purification for FtsZ proteins. Or we can align the gene sequences of the FtsZ proteins we currently have in our regular product line to see which protein is the closest homolog to your FtsZ protein of interest. We recommend using a FtsZ protein that has the closest homology to your target FtsZ protein. While the function of the FtsZ proteins from different bacteria may be the same, there are differences in sequence homology between FtsZ proteins across bacterial lines, strongly suggesting that each FtsZ protein will have a unique pharmacological profile in response to drug treatment. In addition, different FtsZ proteins are likely to have different binding proteins (or at least bind at different affinities) and FtsZ protein dynamics are likely to differ as well. For these reasons, we recommend using FtsZ proteins that match your FtsZ protein of interest in regard to sequence homology.
Question 2: What is the proper way to store the FtsZ protein (Cat. # FTZ01)?
Answer 2: The lyophilized protein should be stored at 4°C with fresh desiccant where it will be stable for 6 months. Storage at -70°C is also an option, though the rubber seal on the container can crack at this temperature and allow in moisture. Therefore we recommend storing at 4°C. If stored at -70°C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized. After reconstituting as directed, the concentrated protein should then be aliquoted and snap frozen in liquid nitrogen. Store these aliquots at -70°C for 6 months. For working concentrations, further dilution of the protein should be made with MES Buffer (100 mM MES pH6.5, 1mM EGTA). NOTE: It is very important to snap freeze the FtsZ protein in liquid nitrogen as other methods of freezing will result in significantly reduced activity. Defrost rapidly by placing in a room temperature water bath for 1 min. Avoid repeated freeze/thaw cycles.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.
