Purification of rabbit sarcoplamic reticulum Ca2+-ATPase expressed in yeast

Author(s)

Eduardo Moraes Rego Reis

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2000-09-12

Examining board members

Sergio Verjovski de Almeida; Sergio Teixeira Ferreira; Angelo Geraldo Gambarini; Fernando de Castro Reinach; Carlos Eduardo Winter

• Advisor: Sergio Verjovski de Almeida

Abstract

We describe in this work a new method for the production of SERCA-l Ca2+-ATPase in yeast using a heat-shock regulated expression vector. Following solubilization of yeast membranes with lysophospholipids, the presence of an hexahistidine tag introduced at the Nterminal end of the Ca2+-ATPase allowed its purification by metal chelating affinity chromatography using a nickel-NTA resin. Using this procedure highly enriched ftactions (75% oftotal protein in the ftaction) of yeast-expressed rabbit Ca2+-ATPase were obtained. Detergent-solubilized 6xHis-Ca2+-ATPase retained highly active (1.5 - 2 µmol/mg protein .min) calcium-dependent, vanadate inhibitable ATPase activity as determined by 32P-γ-ATP hydrolysis. Titration of ATPase activity as a function of ftee calcium revealed high Ca2+ affinity (K0.5 =~ 0.15 µM) and the persistence of a strong cooperative pattem of calcium activation (Hill number of 2.07). The yield and purity of 6xHis Ca2+-ATPase fractions produced with this method allows the biochemical and spectroscopic characterization of Ca2+-ATPase mutants produced in the course of this work. Conversion of the energy present in chemical bonds to electrochemical gradient is a central theme of bioenergetics. It is hoped that the study of the Ca2+-ATPase tryptophan mutants generated in this work will contribute to a better understanding of the coupling mechanism between ATP hydrolysis and the vectorial transport of ions across membranes that occur in this model system. (AU)