Yeast-based production and purification of HIS-tagged human ATAD3A, A specific target of S100B

ATAD3 is a mitochondrial integral inner membrane ATPase with unknown function. ATAD3 is absent in yeast and protozoan and present in all pluricellular eucaryotes where its expression is essential for development. To date, bacterial-based expression of full-length ATAD3 has been unsuccessful because of very high levels of endogenous degradation. Based on Saccharomyces cerevisiae as a heterogeneous expression system, we engineered a high copy strain expressing human ATAD3A-Myc-HIS at a relative high level (2.5 mg/l of yeast culture) without significantly affecting yeast growth. Most of the expressed human ATAD3A-Myc-HIS co-purified with the yeast mitochondrial fraction thus suggesting that targeting to this organelle is preserved in yeast. Like the endogenous protein in human cells, ATAD3A-Myc-HIS expressed in yeast is found resistant to extraction with salt and certain detergents, suggesting membrane insertion. Sarkosyl, C13-DAO, C12-DAO and ONMG efficiently solubilized ATAD3A-Myc-HIS from yeast extracts, but these soluble species did not bind to agarose–nickel matrix. By contrast, urea-denaturated ATAD3A-Myc-HIS bound to agarose–nickel beads and could be renatured and eluted to obtain highly pure ATAD3A-Myc-HIS. As the native protein in vivo, this recombinant, renatured species specifically bound in vitro to S100B and S100A1 in Far-Western assays.

► Many laboratories have had unsuccessful trials in purifying full length ATAD3 in bacteria.
► We succeeded in purifying tagged human ATAD3 in yeast Saccharomyces cerevisiae.
► Human ATAD3, a mitochondrial inner membrane ATPase, reach yeast mitochondria compartment.
► The full length tagged protein was purified efficiently under denaturing/renaturing conditions.
► Purified ATAD3 retains its ability to bind specifically to S100B.