Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

Abstract

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

Structure of the human heterodimeric transporter 4F2hc-LAT2 in complex with Anticalin, an alternative binding protein for applications in single-particle cryo-EM

“…1 b). The minor bands at 60–75 kDa are attributed to glycosylated 4F2hc proteins from HATs that are disrupted by SDS-denaturation and/or during SDS-PAGE 23 , 24 , 38 .

Figure 1 Size-exclusion chromatography (SEC) and SDS-PAGE analysis of the purified human 4F2hc-LAT2::D11vs complex.

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“…To heterologously overexpress human 4F2hc-LAT2, a previously characterized and reported P. pastoris clone was used 12 , 14 , 37 . The recombinant protein was produced in P. pastoris as described 23 , solubilized with GDN and purified according to published protocols 37 , 38 . The anticalin D11vs was produced at preparative scale via secretion in E. coli KS272 with a C-terminal Strep-tag II using the plasmid pNGAL98-D11vs 36 .…”

Characterization of substrates and inhibitors of the human heterodimeric transporter 4F2hc-LAT1 using purified protein and the scintillation proximity radioligand binding assay

“…This construct contains recombinant human 4F2hc-LAT1 bearing N-terminal His- (4F2hc) and Strep- (LAT1) tags. The screening and selection protocol of 4F2hc-LAT1 protein expression clones was detailed in ( Kantipudi et al, 2021 ). Large-scale overexpression in P. pastoris , membrane isolation and protein purification of human 4F2hc-LAT1 using the detergent glyco-diosgenin (GDN, Anatrace) were performed as described in detail in ( Kantipudi et al, 2021 ).…”

“…The methylotrophic yeast Pichia pastoris is a well-established cellular factory for the production of recombinant mammalian membrane proteins ( Byrne, 2015 ; Looser et al, 2015 ; Pochini and Galluccio, 2022 ). Over the years, different human LATs and HATs were successfully overexpressed in P. pastoris for functional and structural studies ( Costa et al, 2013 ; Meury et al, 2014 ; Rosell et al, 2014 ; Jeckelmann and Fotiadis, 2019 ; 2020 ; Kantipudi et al, 2020 ; Kantipudi et al, 2021 ; Kantipudi and Fotiadis, 2021 ; Jeckelmann et al, 2022 ). For example, cell-based transport assays using P. pastoris overexpressing 4F2hc-LAT1 were established and used for the characterization of the specificity for substrates and inhibitors, and their kinetic parameters ( Kantipudi et al, 2020 ; Kantipudi and Fotiadis, 2021 ).…”

“…For example, cell-based transport assays using P. pastoris overexpressing 4F2hc-LAT1 were established and used for the characterization of the specificity for substrates and inhibitors, and their kinetic parameters ( Kantipudi et al, 2020 ; Kantipudi and Fotiadis, 2021 ). Recently, a protocol for the production in P. pastoris and purification of human 4F2hc-LAT1 was reported that allows the isolation of milligram amounts of pure, correctly assembled, stable and properly folded heterodimer ( Kantipudi et al, 2021 ).…”