FtsZ Protein: E. Faecalis Recombinant 6xHis-Tagged
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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.
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.
Purity
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.
Biological activity
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.
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.