Purification and characterization of a putative metalloprotein from Thermotoga maritima.

摘要

Metals are essential to all biological systems and approximately one third of all proteins are associated with a metal. These metal ions are necessary for structure and activity in most of these proteins, but an excess or deficiency of these metal ions can have lethal results. To gain a better understanding of metals in biological systems and the proteins that bind and interact with these metals, four proteins thought to bind metals based on their sequence were chosen from the organism Thermotoga maritima. E. coli cells containing the expression plasmid pMH1 with the gene sequence coding for one of the four proteins of interest were generously supplied by Dr. Scott A. Lesley (Scripps Research Institute, La Jolla, CA). All four proteins of interest were expressed with a hexa-Histidine tag and purified using a heat treatment, followed by a salting out step, and then using an affinity column packed with nickel-sepharose. Only one of the four proteins (TM0440) from the initially selected group was pursued for further characterization due to time restraints. Purified TM0440 was determined to exist as a tetramer in its native state and although initial experiments involving induction with copper supplementation suggested TM0440 might bind with the metal, a spectrophotometric assay outlined in Srinivasan, et al., 1998, showed that TM0440 does not contain a Cu(I)-thiolate binding complex. Further analysis with Inductively-Coupled Plasma Mass Spectroscopy suggests that the protein does not bind copper at all but that it might bind zinc.