title: An improved method for expression and purification of functional human Ca(2+) transporter PMCA4b in Saccharomyces cerevisiae.
abstract: Human plasma membrane calcium ATPases (PMCAs) are highly regulated transporters responsible for the extrusion of calcium out of the cell. Since calcium homeostasis is implicated in several diseases and neurodegenerative disorders, understanding PMCAs activity is crucial. One of the major hindrances is the availability of these proteins for functional and structural analysis. Here, using the yeast Saccharomyces cerevisiae system, we show a new and enhanced method for the expression of the full-length human PMCA isoform 4b (hPMCA4b) and a truncated form lacking its auto-inhibitory domain. We have also improved a method for the purification of the native isoform by calmodulin-agarose affinity chromatography, and developed a new method to purify the truncated isoform by glutathione-Sepharose affinity chromatography. One of the most relevant features of this work is that, when compared to PMCAs purification from pig brain, our method provides a pure single isoform instead of a mixture of isoforms, essential for fine-tuning the activity of PMCA4b. Another relevant feature is that the method described in this work has a superior yield of protein than previously established methods to purify PMCA proteins expressed in yeasts.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas s/n, 06006 Badajoz, Spain.
authors.forename: Isaac
authors.initials: I
authors.lastname: Corbacho
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas s/n, 06006 Badajoz, Spain.
authors.forename: Francisco F
authors.initials: FF
authors.lastname: García-Prieto
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas s/n, 06006 Badajoz, Spain.
authors.forename: Ara E
authors.initials: AE
authors.lastname: Hinojosa
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas s/n, 06006 Badajoz, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Berrocal
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas s/n, 06006 Badajoz, Spain. Electronic address: anam@unex.es.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Mata
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013552:Swine
chemical_list: D011994:Recombinant Proteins; C507517:ATP2B4 protein, human; D053499:Plasma Membrane Calcium-Transporting ATPases
keywords: Calmodulin; Plasma membrane calcium pump; Purification; Yeast; hPMCA4b