FtsZ Protein (Streptococcus pneumoniae recombinant plus 6xHis tag)

On Arrival: 4°C

Question 1: Given the high sequence homology between FtsZ proteins, is it necessary to use 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. #FTZ03)?

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.

Identification and characterization of FtsZ binding proteins
Characterization of FtsZ dynamics
Developing FtsZ ligands that may be used as anti-bacterial agents.

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 ControlWhen 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 ControlWhen a specific activity greater than 0.05 mol GTP / mol FtsZ / min is obtained in Protofilament Buffer.

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