Western Kentucky University

Poster Title

Novel Purification Protocol for Heparin Binding Proteins - Relevance in Biopharmaceuticals

Institution

Western Kentucky University

Abstract

Heparin binding proteins including fibroblast growth factors (FGF’s) and its receptors mediate a wide range of important cellular processes, which makes this class of proteins biomedically important. These proteins also play a significant role in various stages of development, morphogenesis, angiogenesis, and wound healing processes. Engineering heparin binding proteins can bring many advantages, but it requires cost effective and efficient purification methodologies compared to the currently available methods. In this context, in the present study, we report an efficient off-column purification of FGF-1 from soluble fractions as well as purification of protein which expresses as insoluble inclusion bodies like D2 domain of FGFR using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which has several disadvantages and resulted in higher cost for production of recombinant proteins. Human FGF-1 and the D2 domain have been expressed in Escherichia coli in high yields. The proteins were purified to homogeneity (~98% purity) using IRC resin. Results of the heparin binding affinity chromatography and steady state fluorescence experiments showed that the recombinant FGF-1 and the D2 were in a native and biologically active conformation. The findings of the present study not only pave the way for an in depth structural-functional investigation of this class of proteins but also provide avenues for efficient and inexpensive purification of other biopharmaceutically important biological macromolecules.

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Novel Purification Protocol for Heparin Binding Proteins - Relevance in Biopharmaceuticals

Heparin binding proteins including fibroblast growth factors (FGF’s) and its receptors mediate a wide range of important cellular processes, which makes this class of proteins biomedically important. These proteins also play a significant role in various stages of development, morphogenesis, angiogenesis, and wound healing processes. Engineering heparin binding proteins can bring many advantages, but it requires cost effective and efficient purification methodologies compared to the currently available methods. In this context, in the present study, we report an efficient off-column purification of FGF-1 from soluble fractions as well as purification of protein which expresses as insoluble inclusion bodies like D2 domain of FGFR using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which has several disadvantages and resulted in higher cost for production of recombinant proteins. Human FGF-1 and the D2 domain have been expressed in Escherichia coli in high yields. The proteins were purified to homogeneity (~98% purity) using IRC resin. Results of the heparin binding affinity chromatography and steady state fluorescence experiments showed that the recombinant FGF-1 and the D2 were in a native and biologically active conformation. The findings of the present study not only pave the way for an in depth structural-functional investigation of this class of proteins but also provide avenues for efficient and inexpensive purification of other biopharmaceutically important biological macromolecules.