Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris |
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| Authors: | Meritxell Costa Albert Rosell Elena Álvarez-Marimon Antonio Zorzano Dimitrios Fotiadis Manuel Palacín |
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| Institution: | 1. Institute for Research in Biomedicine (IRB Barcelona), Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, E-08028 Barcelona, Spain;2. Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona, Spain;3. Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, CH-3012 Bern, Switzerland |
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| 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 ～1 mg of HAT from 2 l 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. |
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