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dc.contributor.authorSuttisansanee, Uthaiwanen 14:07:19 (GMT) 14:07:19 (GMT)
dc.description.abstractBacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.en
dc.format.extent3748786 bytes
dc.publisherUniversity of Waterlooen
dc.rightsCopyright: 2006, Suttisansanee, Uthaiwan. All rights reserved.en
dc.subjectHexahistidine-affinity tagen
dc.subjectNickel-nitrilotriacetic aciden
dc.subjectdynamic light scatteringen
dc.subjectgel filtration chromatographyen
dc.subjectfluorescence measurementen
dc.titleBiochemistry in Bacterioferritinen
dc.typeMaster Thesisen
dc.pendingfalseen and Biochemistryen
uws-etd.degreeMaster of Scienceen

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