Innovative Purification Protocol for Heparin Binding Proteins: Relevance in Biopharmaceutical and Biomedical Applications

<p>Heparin binding (HB) proteins mediates a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins could bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to the currently available methods. One of the most important classes of heparin binding protein is the fibroblast growth factors (FGFs) and its receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which exhibit several disadvantages, including time-consuming experimental procedures and regeneration and results in high cost for production of recombinant proteins. Authenticity of the purified proteins was verified by SDS-PAGE and MALDI mass spectrum analysis. Results of the heparin binding chromatography and steady state fluorescence experiments showed that the FGF-1 and the D2 are in a native biologically active conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.</p>

Name thesis:degree_name

Master of Science

Discipline thesis:degree_discipline

Department of Chemistry

Year

2011

Author dc:creator

• Batra, Sumit

Contributors dc:contributor

• Dr. Rajalingam Dakshinamurthy (Director), Dr. Eric Conte, Dr. Chad Snyder

• affinity chromatography
• thermal denaturation
• biological activity
• fibroblast growth factor
• protein purification
• Chemistry
• Medicinal and Pharmaceutical Chemistry

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/1062

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-2065