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URL: http://www.addgene.org/103001
Proper Citation: RRID:Addgene_103001
Insert Name: PfSSB_W166C
Organism: Other
Bacterial Resistance: Ampicillin
Defining Citation: PMID:29466468
Vector Backbone Description: Vector Backbone:pET22B; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Comments: Usage: The expressed protein is a Plasmodium falciparum single-stranded DNA-binding protein, PfSSB, with two point mutations, C93A and W166C, for subsequent labeling at the 166 cysteine with one IDCC (N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide) or Cy3B per subunit. PfSSB was expressed without the N-terminal apicoplast localisation sequence (76 amino acids) but amino acids are numbered for the full-length protein, that is the first amino acid in the expressed protein is M77.W166C is the equivalent position to W88 in E. coli SSB, which was labeled with Cy3B for single molecule measurements of DNA unwinding using TIRF (Fili, N.; Mashanov, G.; Toseland, C. P.; Batters, C.; Wallace, M. I.; Yeeles, J. T. P.; Dillingham, M. S.; Webb, M. R.; Molloy, J. E., Visualizing DNA unwinding by helicases at the single molecule level. Nucleic Acids Res. 2010, 38, 4448-4457.) Protocol: See Hedgethorne and Webb (2012), which is also followed for PfSSB purification, except with the omission of the PEI precipitation step. Hedgethorne, K.; Webb, M. R., Fluorescent SSB as a reagentless biosensor for single-stranded DNA. Methods Mol. Biol. 2012, 922, 219-233.
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Source: Addgene