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Axial Ligand Mutant: H229A

Many pathogenic bacteria use their iron acquisition mechanisms to live inside hosts. Streptococcus pyogenes is a pathogenic bacterium that uses streptococcal iron acquisition ABC transporter to obtain heme. SiaA (HtsA, spy1795), a lipoprotein located on the cell surface, serves as a heme binding protein. To understand the iron-uptake mechanism, histidine 229, one of the two proposed axial ligands in SiaA, was mutated to alanine. SiaA H229A was expressed in E. coli, lysed by French Press, and purified by fast protein liquid chromatography (FPLC). SDS-PAGE indicated that pure protein was isolated. Nickel affinity FPLC gave purer H229A when 0.5 M imidazole was added to the binding buffer. Overall, histidine 229 is likely to be an axial ligand in wild type SiaA, as shown by the fact the mutant readily lost heme as evidenced by UV-vis spectra.

Identiferoai:union.ndltd.org:GEORGIA/oai:digitalarchive.gsu.edu:honors_theses-1000
Date08 August 2008
CreatorsNguyen, Nhung Phuong
PublisherDigital Archive @ GSU
Source SetsGeorgia State University
Detected LanguageEnglish
Typetext
Formatapplication/pdf
SourceUndergraduate Honors Theses

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