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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.
Entercoccus faecalis FtsZ protein has been purified after over-expression in E.coli. E.f. FtsZ has a 6xHis tag and an approximate molecular weight of 55 kDa (Fig. 1). FTZ04 is provided as a lyophilized protein with buffer and stabilizers.
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 10% polyacrylamide gel. FtsZ protein was calculated to be >90% pure (see Figure 1, Lane 2).
Figure 1: A 100 µg sample of E.f. FtsZ protein was separated on a 4-20% gradient SDS-PAGE gel and stained with Coomassie blue. Lane 1, 100µg FTZ04. SeeBlue molecular weight markers are from Invitrogen.
The biological activity of FtsZ can be determined in two ways, first from its ability to efficiently polymerize into protofilaments and sheets in vitro in the presence of Mg2+ and GTP, and secondly to hydrolyze GTP to GDP and Pi. Polymerization with Mg2+:GTP in the presence of a crowding reagent (e.g. carbohydrate polymers Ficoll or dextran in "Sheet Buffer") results in sheets of FtsZ protofilaments that sediment at 100,000xg, analysis of the pellet and sup by protein assay or SDS-PAGE indicates the proportion of polymerizable FtsZ protein; the batch of protein passes Quality Control when the pelleted amount is greater than 60% of the total and the supernatnat protein is in agreement with its known critical concentration for assembly. GTPase activity is determined in a "Protofilament Buffer" by measuring the Pi released over time by using the MESEG phosphate detection kit (Cat# BK052) or an endpoint assay (Cat. # BK054). The batch of protein passes Quality Control when a specific activity greater than 0.05 mol GTP / mol FtsZ / min is obtained in Protofilament Buffer.
Question 1: Given the high sequence homology between FtsZ proteins, why does using a species-specific FtsZ matter?
Answer 1: FtsZ sequence homology between some species is high e.g. S.enterica and E.coli, while most others have 40-70% homology. Even with high homology, if the divergent sequences encode a binding domain for FtsZ accessory proteins or novel compounds/drugs, binding of the proteins and/or test compounds could vary significantly. Whether using FtsZ as a drug discovery tool for novel antibiotic research or for better understanding bacterial cell division, it is best to use FtsZ proteins that are specific for the bacterial species you are studying to insure reliable and consistent results.
Question 2: What is the proper way to store the FtsZ protein (Cat. # FTZ04)?
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.
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