Purification and preliminary structural characterization by NMR spectroscopy of the "HoLaMa" DNA polymerase

HoLaMa is a Klenow sub-fragment lacking the 3’-5’ exonuclease domain, whose gene codes for residues 515-928 of Escherichia coli DNA polymerase I. The enzyme was designed in a previous study starting from different Klenow enzymes, with the aim of studying a mini-DNA polymerase with NMR spectroscopy. In the present work, we studied a new purification protocol for the production of HoLaMa in order to obtain an appropriate quantity for NMR analysis. We tested three different purification procedure and at the end, we collected 5.8 mg of HoLaMa (Volume 1.8 mL, concentration 67 μM). After the purification, we started the study of HoLaMa by NMR spectroscopy, focusing on the nature of enzyme-substrate interactions and studying the kinetics of the reaction. Our preliminary studies were designed to understand the characteristic NMR signals of HoLaMa under different conditions of temperature and buffer; finally, we also focused our analysis on the interactions between protein, DNA and nucleotides.