Date of Graduation

12-2011

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

Henry, Ralph L.

Second Committee Member

Hestekin, Christa N.

Third Committee Member

Kim, Jin-Woo

Keywords

Applied sciences; Biological sciences; Affinity tails; Chromatography; Protein purification

Abstract

At industrial scale, reducing the step in purification and recovery is desired; this not only decreases the cost but also increases the yield. Hydrophobic Interaction Chromatography (HIC) and Immobilized Metal Affinity Chromatography (IMAC) both are not harsh on biological structure or activity of proteins; also both the techniques are economical and therefore a suitable choice at industrial level. This dissertation comprises of three parts. Purpose for the Part I was to identify and characterize Escherichia coli proteins which display affinity towards both IMAC and Hydrophobic Interaction Chromatography (HIC). Co (II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Nine out of the ten proteins identified have iso-electric values less than six, and half are considered nonessential. These data indicated that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC - HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome. Part II comprises of study of E. coli Alpha Galactosidase (MelA) as a candidate for affinity tag for this dual step purification approach. Affinity of MelA towards Co (II) IMAC and HIC was confirmed by fusing with a small peptide. Metal binding motif and various salt binding motifs of MelA were identified and studied. A MelA based affinity tag NT1 was developed by identifying the major salt binding region for HIC. It is desirable to have a tag which doesn't interfere with solubility or biological activity of the fused protein. Solubility has been the biggest issue with the development of affinity tag for HIC. In part III, with successful use of novel proteome, we report development of MelA as a successful affinity tag for HIC especially as it does not affect the expression, solubility or biological activity of the fused protein, with very few co-eluting proteins as contaminants.

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