title: Yeast-based production and purification of HIS-tagged human ATAD3A, A specific target of S100B.
abstract: 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 (2.5mg/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.
authors.affiliation: Université Joseph Fourier, Laboratoire de Bioénergétique Fondamentale et Appliquée, BP53 38041 Grenoble Cedex, France.
authors.forename: Shuijie
authors.initials: S
authors.lastname: Li
authors.forename: Benjamin
authors.initials: B
authors.lastname: Clémençon
authors.forename: Patrice
authors.initials: P
authors.lastname: Catty
authors.forename: Gérard
authors.initials: G
authors.lastname: Brandolin
authors.forename: Uwe
authors.initials: U
authors.lastname: Schlattner
authors.forename: Denis
authors.initials: D
authors.lastname: Rousseau
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D026481:Blotting, Far-Western; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009414:Nerve Growth Factors / Q000378:metabolism*; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014508:Urea / Q000378:metabolism
chemical_list: C549686:ATAD3A protein, human; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C110294:S100A1 protein; C579160:S100B protein, human; D006639:Histidine; D014508:Urea; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities