The cysteine-free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications

Abstract

APEX2, an engineered ascorbate peroxidase for high activity, is a powerful tool for proximity labeling applications. Owing to its lack of disulfides and the calcium-independent activity, APEX2 can be applied intracellularly for targeted electron microscopy imaging or interactome mapping when fusing to a protein of interest. However, APEX2 fusion is often deleterious to the protein expression, which seriously hampers its wide utility. This problem is especially compelling when APEX2 is fused to structurally delicate proteins, such as multi-pass membrane proteins. In this study, we found that a cysteine-free single mutant C32S of APEX2 dramatically improved the expression of fusion proteins in mammalian cells without compromising the enzyme activity. We fused APEX2 and APEX2^C32S to four multi-transmembrane solute carriers (SLCs), SLC1A5, SLC6A5, SLC6A14, and SLC7A1, and compared their expressions in stable HEK293T cell lines. Except the SLC6A5 fusions expressing at decent levels for both APEX2 (70%) and APEX2^C32S (73%), other three SLC proteins showed significantly better expression when fusing to APEX2^C32S (69 ± 13%) than APEX2 (29 ± 15%). Immunofluorescence and western blot experiments showed correct plasma membrane localization and strong proximity labeling efficiency in all four SLC-APEX2^C32S cells. Enzyme kinetic experiments revealed that APEX2 and APEX2^C32S have comparable activities in terms of oxidizing guaiacol. Overall, we believe APEX2^C32S is a superior fusion tag to APEX2 for proximity labeling applications, especially when mismatched disulfide bonding or poor expression is a concern.

Keywords: APEX2; ascorbate peroxidase; proximity labeling; solute carriers.

Figure 1

Crystal structures of ascorbate peroxidase in complex with (a) ascorbate (PDB: 1OAF) and (b) salicylhydroxamic acid (PDB: 1V0H) showed distinct binding sites of ascorbate and small aromatic molecules. Spheres: Cys‐32; magenta: heme; cyan: ascorbate in (a) or salicylhydroxamic acid in (b)

Figure 2

APEX2 and APEX2^C32S showed comparable activity of guaiacol oxidation. (a) Initial rates of guaiacol oxidation (V_o) were measured in a range of H₂O₂ concentrations for APEX2 and APEX2^C32S. A fixed concentration of guaiacol at 0.735 mM was used. (b) Kinetic measurements of guaiacol oxidation using APEX2 and APEX2^C32S. A fixed concentration of H₂O₂ at 1 mM was used. For clarity, only three guaiacol concentrations are shown. (c) The Lineweaver–Burk (double reciprocal) plot of APEX2 and APEX2^C32S enzyme kinetics. (d) The Michaelis–Menton parameters of APEX2 and APEX2^C32S

Figure 3

Expression level and subcellular localization of SLC‐EGFP fusions in HEK293T cells. (a) Expression levels of SLC‐EGFP fusions analyzed by flow cytometry. Red: SLC‐fusion; gray: parental HEK293T. (b) Sub‐localization of SLC‐EGFP fusions analyzed by confocal microscope. All SLC‐EGFP fusions were detected by EGFP fluorescence. EGFP, enhanced green fluorescence protein; SLC, solute carriers

Figure 4

The expression levels of SLC‐APEX2^C32S are generally better than SLC‐APEX2 in HEK293T cells. (a) Flow cytometry experiments compare the expressions levels of four SLC‐APEX2^C32S and SLC‐APEX2 constructs. Red: SLC‐fusion; gray: parental HEK293T. (b) Immunofluorescence experiments show the correct plasma membrane localization of SLC‐APEX2^C32S and SLC‐APEX2 proteins. All SLC‐APEX2 and SLC‐APEX2^C32S fusions were detected by Alexa647 Myc‐tag staining. SLC, solute carriers

Figure 5

SLC‐APEX2^C32S exhibited significant proximity labeling efficiency using biotin tyramide. (a) The proximity labeling efficiency was analyzed by flow cytometry for four SLC‐APEX2^C32S constructs in HEK293T cells. Red: both biotin tyramide and H₂O₂ were added; gray: only H₂O₂ was added. (b) Immunofluorescence analyses of SLC‐APEX2^C32S proximity labeling. APEX2^C32S was detected by Myc‐tag staining (red). The biotin tyramide labeling was detected by streptavidin (green). SLC, solute carriers

Figure 6

SLC1A5‐APEX2 proximity labeling is superior to SLC1A5‐APEX2^C32S in HEK293T cells. (a) The expression level (upper) and the proximity labeling efficiency (bottom) of SLC1A5‐APEX2 and SLC1A5‐APEX2^C32S were analyzed by flow cytometry. Upper: Myc‐tag staining for expression level (red: SLC1A5‐APEX2 or SLC1A5‐APEX2^C32S cells; gray: parental HEK293T). Bottom: streptavidin staining for proximity labeling efficiency (red: both biotin tyramide and H₂O₂ were added; gray: only H₂O₂ was added). (b) The proximity labeling efficiency was analyzed by western blots. The red arrows denote the bands that are clearly observed in SLC1A5‐APEX2^C32S, but very vague in SLC1A5‐APEX2. SLC, solute carriers