Show simple item record

dc.contributor.advisorFishel, Richard
dc.creatorBennett, Jared
dc.description.abstractSingle molecule visualization studies require proteins that are labeled with known fluorophores at known locations on the molecule. This project examines the viability of one method that is capable of specifically and efficiently labeling proteins with fluorophores. The labeling method uses the Formylglycine Generating Enzyme (FGE) that recognizes a hexa-amino acid sequence and catalyzes the conversion of a central cysteine into a formyl-glycine containing a reactive aldehyde group. The converted aldehyde may be used in conjunction with Hydrazino-Pictet-Spengler (HIPS) ligation to attach a high quantum yield fluorophore such as Cy3 or Cy5 to a specific site within any protein. We used MutS as an initial test of this method. The fusion cloning method was then extended to the eukaryote PCNA trimer. We then cloned and modified the RecJ protein to more fully test the labeling methodology. RecJ is a 5'-3' exonuclease that functions in conjunction with MutS and MutL to execute bacterial MMR. To date, no exonuclease has been labeled and visualized for single molecule analysis. Finally, in order to test whether fluorophore-labeling RecJ affected its activity, we developed a novel fluorophore-based exonuclease assay.en_US
dc.publisherThe Ohio State Universityen_US
dc.relation.ispartofseriesThe Ohio State University. Department of Biochemistry Honors Theses; 2014en_US
dc.subjectMismatch Repairen_US
dc.subjectFormylglycine Generating Enzyme (FGE)en_US
dc.subjectSingle-Molecule Analysisen_US
dc.titleGeneration of Mismatch Repair Fusion Proteins for Single-Molecule Analysisen_US
dc.description.embargoNo embargoen_US
dc.description.academicmajorAcademic Major: Biochemistryen_US

Files in this item


Items in Knowledge Bank are protected by copyright, with all rights reserved, unless otherwise indicated.

This item appears in the following Collection(s)

Show simple item record