title: Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris.
abstract: Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ~1mg of HAT from 2l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, E-08028 Barcelona, Spain.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Costa
authors.forename: Albert
authors.initials: A
authors.lastname: Rosell
authors.forename: Elena
authors.initials: E
authors.lastname: Álvarez-Marimon
authors.forename: Antonio
authors.initials: A
authors.lastname: Zorzano
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
authors.forename: Manuel
authors.initials: M
authors.lastname: Palacín
mesh_terms: D002846:Chromatography, Affinity; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000096:biosynthesis* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D007930:Leucine / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D027282:Fusion Regulatory Protein 1, Heavy Chain; D011994:Recombinant Proteins; D007930:Leucine