Date of Graduation

8-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Thallapuranam Kumar

Committee Member

Roger Koeppe

Second Committee Member

Jingyi Chen

Third Committee Member

Dan Davis

Keywords

Affinity Tag, Heparin, Heparin-binding Proteins

Abstract

Heparin is a polydispersed sulfated molecule that is part of the family called glycosaminoglycans found in the extracellular matrix and cell surfaces. This molecule is extremely important for the activation of proteins and protein-receptor interactions that are responsible for downstream cell signaling pathways. Heparin has been isolated from porcine intestine and used as an anticoagulant for the prevention of embolisms, heart thrombosis, and clotting during heart surgeries. This so-called miracle drug was in use until 2008, when isolated batches were found to be contaminated with other glycosaminoglycans similar to heparin. From 2008, there has been a dire need for a more cost-effective purification of heparin in good yield, to remove the contaminants.

Heparin has also been used for affinity chromatography to purify heparin-binding proteins. Recombinant protein purification is an important aspect of biotechnology, with a heavy emphasis on developing easy purification systems for large quantities of homogeneously pure therapeutic proteins at low costs. Several affinity tags have been employed to aid in the expression and purification of various proteins. There are, however, various disadvantages, such as interference with the target protein structure, unsuitable elution conditions, multiple-step purification procedures, and many others.

In this context, a novel heparin-binding peptide (HB-peptide) was designed based on the heparin-binding region of acidic fibroblast growth factor (FGF-1). The HB-peptide has been shown to bind to heparin with better affinity than other glycosaminoglycans. A one-step Heparin-Sepharose-based purification procedure was developed for various recombinant proteins using mild elution conditions. Using proteolytic cleavage, the affinity tag was removed to obtain a folded protein. Polyclonal antibodies were also grown against the specific sequence of the HB-peptide, which then were used to successfully and specifically bind fusion proteins that only contained the HB-peptide even at the nanogram level.

HB-peptide was then coupled onto a solid matrix to be used as an affinity column, in a similar fashion as heparin-Sepharose with HBPs. A mixture of glycosaminoglycans were separated from each other for the most part. In this context, our HB-peptide can be used as a means to separate glycosaminoglycans, yielding pure heparin for future medical use.

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