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

Kantipudi, Satish; Harder, Daniel; Bonetti, Sara; Fotiadis, Dimitrios; Jeckelmann, Jean-Marc (2021). Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein. Methods and protocols, 4(3) MDPI AG 10.3390/mps4030051

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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.

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