Expression, Purification, and Characterization of the SIAA M79A Protein

• Main Author: Basden, Brian
• Format: Others
• Published: Digital Archive @ GSU 2007
• Subjects: Heme; SiaA; Streptococcus pyogenes; heme binding proteins; Axial ligand; Methionine; Alanine; Chemistry
• Online Access: http://digitalarchive.gsu.edu/chemistry_hontheses/1; http://digitalarchive.gsu.edu/cgi/viewcontent.cgi?article=1000&context=chemistry_hontheses

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.