Design of Novel Methods to Eliminate DNA Binding to Recombinant Proteins

Abstract

Successful recovery of pure recombinant proteins after expression and purification is a constant challenge in molecular biology. Along with the need for purity there is also a need for high yield recovery. We have determined another challenge to pure recombinant protein expression in that proteins with high isoelectric points can bind to host bacterial DNA. We have demonstrated the presence of the protein/DNA complex using fluorescence spectroscopy. The focus of this study is to develop expression and purification methods to eliminate host bacterial nucleotide contamination. Data will be presented, using proteins with varying pI values, namely, C2B, FGF-2 , and FGF-1 on the presence and elimination of this DNA/protein complex. Studies will be presented on the effect of pH, salt, DNAase, and phospholipase on eliminating contaminant bound DNA. The effect of the host bacterial contaminant bound DNA on the structure and function of recombinant proteins will be assessed using various biophysical techniques, including multidimensional NMR spectroscopy.