Date of Graduation

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Beitle, Robert R. Jr.

Committee Member

Durdik, Jeannine M.

Second Committee Member

Kim, Jin-Woo

Third Committee Member

Goforth, Robyn L.

Keywords

E. coli; Immobilized Metal Affinity Chromatography; Chromosomal mapping

Abstract

The dissertation is comprised of three parts. Part I describes proteomic analysis of native bacterial proteins from Escherichia coli (E.coli) that bind during Immobilized Metal Affinity Chromatography (IMAC). Part II describes the value in exploiting proteome based data as a tool toward the design an E. coli expression strain that is particularly useful when Immobilized Metal Affinity Chromatography is employed as the initial capture step of a homologous protein purification process. Part III describes a methodology of chromosomal mapping of all contaminant gene products.

The objective of Part I was to identify all E. coli proteins that bind to Co(II), Ni(II), and Zn(II) IMAC columns, describing the isoelectric point, molecular weight, and metabolic essentiality of the characterized proteins were considered. Information regarding this group of proteins is presented and used to define the IMAC bioseparation-specific metalloproteome of E. coli. Such data concerning the potential contaminant pool is useful for the design of separation schemes, as well as designing optimized affinity tails and strains for IMAC purification. Part II examined proteins known to co-elute during Co(II), Ni(II), and Zn(II) IMAC purifications. Methods to circumvent the effects of punitive protein removal were proposed and carried out. Specifically, triosephosphate isomerase (TIM; tpiA gene product), a protein known to bind during IMAC, was redesigned through site directed mutagenesis to eliminate surface exposed histidine. By extension of this rational, Part III provides a theoretical model of using in silico mapping (Circos diagrams) to create a practical system of applying data described Part I. Such a tool has potential to allow future investigators the possibility of mapping large scale genomic deletions; significantly streamlining cell line development when compared to the individual targeting methodologies seen in Part II.

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