The Use Of High-Field Nmr To Determine Homogeneity In A Preparation Of Human C5a Produced By Escherichia-Coli

The polypeptide backbone of human C5a was prepared by recombinant DNA techniques. Standard biochemical analysis guided the protein separation to give a sample of C5a which was deemed homogeneous. However, nuclear magnetic resonance (NMR) studies showed the material to be significantly heterogeneous. Reanalysis by high performance liquid chromatography (HPLC) corroborated the NMR results. Further separation by HPLC and analysis by NMR spectroscopy guided the isolation of rC5a to greater than 92% purity. NMR analysis, immunochemical and biological evaluation of the impurities showed them to be C5a structural variants. These results indicate that conventional methods of protein chemistry can fail to reveal heterogeneity in recombinant proteins, and in some circumstances NMR spectroscopy can aid in their purification.