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

详细信息    查看全文

• 作者：Meritxell Costa^a ; ^b ; ^c ; ¹ ; Albert Rosell^a ; ^b ; ¹ ; Elena Á ; lvarez-Marimon^a ; ^b ; Antonio Zorzano^a ; ^b ; Dimitrios Fotiadis^c ; ² ; ^{dimitrios.fotiadis@ibmm.unibe.ch} ; Manuel Palací ; n^a ; ^b ; ² ; ^{manuel.palacin@irbbarcelona.org}
• 关键词：4F2hc ; Heteromeric amino acid transporter ; LAT2 ; Membrane protein ; Overexpression ; Pichia pastoris
• 刊名：Protein Expression and Purification
• 出版年：2013
• 出版时间：January, 2013
• 年：2013
• 卷：87
• 期：1
• 页码：35-40
• 全文大小：479 K

文摘

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.