Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

ABSTRACT: Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 μm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2–5 μm) allowed not only to achieve high viable cell concentrations (VCC, 16.4–20.6×10⁶ cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×10⁹ TCID₅₀/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×10₆ cells/mL and infectious virus titers up to 7.1×10¹⁰ TCID₅₀/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level

TEM and Nanogold labelling of purified human 4F2hc-LAT2 heterodimers.

<p>(A) Electron micrograph of purified negatively-stained 4F2hc-LAT2 heterodimers. Galleries of unlabelled (B) and 5 nm Ni-NTA-Nanogold labelled (C) 4F2hc-LAT2 heterodimers. Bilobed particles with two different sized domains are clearly visible. The 5 nm Ni-NTA Nanogold spheres are localized to the larger domains of the heterodimer. The scale bar in (A) represents 50 nm. The frame size of the boxes in (B) and (C) represent 28 nm.</p

3D reconstruction of human 4F2hc-LAT2 purified in DDM, LMNG and CHS.

<p>(A) Side views of the 3D map calculated from 27'921 projections of negatively-stained heterodimer particles (initially 28'993 picked). 4F2hc-LAT2 has a bilobed structure and consists of a smaller tilted density located on top of a larger density (tilt indicated by black dotted lines). As a consequence of the tilt, the 3D map features a distinct cavity (arrowheads) and on the opposite side a seal (marked by a white, dotted curve). Based on the Fourier shell correlation curve (0.5 criterion) the resolution is 20 Å (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109882#pone.0109882.s002" target="_blank">Fig. S2C</a>). (B) and (C) Different views of the 4F2hc-LAT2 3D map with the fitted crystal structure of the 4F2hc-ED (PDB ID: 2DH2). Asterisks in (B) and (C) indicate the location of the N-terminus in the 4F2hc-ED crystal structure. The 4F2hc-ED structure is represented as cartoon (α-helices and β-strands in red and blue, respectively) and surface (CPK colours) models. The scale bar represents 50 Å.</p

SDS-PAGE, Western blot analysis, SEC and SPA of 4F2hc-LAT2 purified in DDM, LMNG and CHS.

<p>(A) Coomassie Blue-stained 10% SDS/polyacrylamide gel (lane C) of 4F2hc-LAT2 after the two affinity chromatography steps. The complex runs as a prominent band at ∼125 kDa. Western blot analysis using anti-4F2hc (lane α4F2_ED) and anti-StrepTagII (lane αSTag) antibodies indicated the presence of intact heterodimers only, i.e., no 4F2hc or LAT2 from disrupted complexes. (B) SEC of purified 4F2hc-LAT2 indicated a prominent almost symmetrical elution peak at 13.7 ml. (C) Silver-stained 10% SDS/polyacrylamide gel of purified 4F2hc-LAT2 after gel filtration (lane ST; from peak fraction). Again, one single band is visible corresponding to the heterodimer. Integrity of the complex was further supported by Western blotting using anti-4F2hc (lane α4F2_ED) and anti-StrepTagII (lane αSTag) antibodies. (D) Radioligand-binding assay by SPA using purified 4F2hc-LAT2 and [³H]L-leucine. Bar 1: Binding of the radiolabelled substrate L-leucine to 4F2hc-LAT2, which is bound to scintillation beads, induces SPA signal. As expected, SPA signal was abolished by addition of 4 mM cold L-leucine (bar 2; competitive inhibition) or 100 mM imidazole (bar 3; detachment of the protein from the SPA beads). Bars represent mean ± SEM from triplicates. One representative of three similar independent experiments is shown.</p