Characterization of a PIB copper transport ATPase from a mesophilic bacteria expressed in yeast.

BOSICH, W; BREDESTON, LM; GONZÁLEZ FLECHA, FL

Villa Carlos Paz

Congreso; XLII reunion anual de la Sociedad Argentina de Biofisica; 2013

Sociedad Aegentina de Biofísica

Cu+ transporting ATPases are integral membrane proteins that have an important role in cooper homeostasis and detoxification. Here we report the production of CopA from Legionella pneumophila (LpCopA) in Saccharomyces cerevisiae using a galactose induced system and GFP as an indicator. We optimized the expression conditions of LpCopA measuring fluorescence in whole cells. Maximal fluorescence was obtained growing the transformed cells in media containing 1% yeast extract, 1% peptone, 0.1% glucose and induced with 1% galactose for 36 hs at 28oC. The GFP technology was critical for monitoring the production and purification steps of this protein, and can be an useful tool for other membrane proteins. The ATPase activity of the purified enzyme was determined as the initial rate of Pi release using a colorimetric method based in the formation of a phosphate-malachite green complex, optimized in our laboratory. The LpCopA ATPase activity was similar to that previously reported (Gourdon, et. Al 2012), but was found to be only dependent on ATP, Mg2+ Cu+, DTT and NaCl concentration.