biostudies-literatureHuang MSMinistry of Science and TechnologyAcademia Sinica1703-1712https://www.ebi.ac.uk/biostudies/studies/S-EPMC6699085biostudies-literatureUnknown28(9)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 tProtein science : a publication of the Protein SocietyThe cysteine-free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications.PMC6699085107‐2113‐M‐001‐013AS‐CDA‐108‐L07Lin WCCheng CHChang JHHuang MSMou KYWang HYfalseThe cysteine-free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications.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 t2019-01-01T00:00:00Z2019 Sep2026-05-02T21:49:11.817Z2020-09-05T07:15:18ZS-EPMC66990853130651610.1002/pro.3685