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The cysteine‐free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications
Author(s) -
Huang MengSen,
Lin WenChing,
Chang JenHsuan,
Cheng ChengHung,
Wang Han Ying,
Mou Kurt Yun
Publication year - 2019
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3685
Subject(s) - fusion protein , chemistry , western blot , interactome , microbiology and biotechnology , cysteine , mutant , biochemistry , hek 293 cells , lipid bilayer fusion , enzyme , biology , membrane , recombinant dna , gene
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.