Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Costa, Meritxell; Rosell, Albert; Álvarez-Marimon, Elena; Zorzano, Antonio; Fotiadis, Dimitrios; Palacín, Manuel (2013). Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris. Protein expression and purification, 87(1), pp. 35-40. Elsevier 10.1016/j.pep.2012.10.003

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Costa Torres, Meritxell and Fotiadis, Dimitrios José

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

1096-0279

Publisher:

Elsevier

Language:

English

Submitter:

Patrizia Catucci

Date Deposited:

01 May 2014 13:00

Last Modified:

01 May 2014 13:00

Publisher DOI:

10.1016/j.pep.2012.10.003

PubMed ID:

23085088

URI:

https://boris.unibe.ch/id/eprint/43407

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