Streamlined purification and characterization of Pyrococcus furiosus rubredoxins with different N-terminal...

Rubredoxins (Rds), like those from Pyrococcus furious (Pf), have largely been found to be expressed in Escherichia coli (E. coli) as a mixture of different N-terminal forms, which may affect the properties of the protein. The typical procedures for the purification of Rds are cumbersome and usually with low yield. We present herein a streamlined purification strategy based on the reversed-phase high performance liquid chromatography (RP-HPLC), which offers high yield and high resolution after simply one-step purification following pre-treatment. We also show that RP-HPLC can be a valuable tool to gain information related to the thermal decomposition pathway of Pf-Rds.