Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment

Structure/function studies of the NAD+-dependent DNA ligase from the poly-extremophile Deinococcus radiodurans reveal importance of the BRCT domain for DNA binding

“…The genes were cloned into the expression vector pDest14 according to the GATEWAY cloning system (GE Healthcare) guidelines. Protein expression experiments were performed according to the small-, medium-, and large-scale expression protocols as described previously for other D. radiodurans enzymes (Fernandes et al 2021 ). The best conditions for expressing DrLigA were at 37 °C for 3 h with the E. coli strain BL21(DE3)* pRARE2.…”

“…Therefore, in large-scale expression of the DrLigA and DrLigAΔBRCT, proteins were expressed according to these conditions and expression was induced with the addition of 0.5 mM of isopropyl β-D-1-thiogalactopyranoside. The initial purification procedure of both full-length and truncated proteins followed three steps: (i) immobilization on metal ion affinity chromatography (IMAC) with a 5 mL HisTrap HP column, (ii) followed by TEV protease cleavage of the His 6 -tag and a (iii) subsequent second IMAC step to remove the tag (Fernandes et al 2021 ). The final purification step was performed using a 1 mL HiTrap Heparin HP column.…”

“…For the additives’ experiments, an 18 µL mix was prepared, and 2 µL solution of additive (300 µM NAD + , 1 mM Mg 2+ , 1 mM Mn 2+ , or 1 mM Zn 2+ ) was added. Fluorescence was measured each minute over a range of temperatures from 25 to 90 °C, as previously described by Fernandes et al ( 2021 ). The peak minimum of the first derivative of Relative Fluorescence Units over temperature was determined as the melting temperature (T m ).…”

“…T h e ge n e -e n c o d i n g D r L i g A ( D R _ 2 0 6 9 ) a n d DrLigAΔBRCT were amplified according to the PCR reactions described by Fernandes et al (2021). For DrLigA amplification, the primers FPDrLigA and RPDrLigA were used in the first PCR reaction.…”