biostudies-literatureSingh AKNarodowe Centrum Nauki125https://www.ebi.ac.uk/biostudies/studies/S-EPMC10334657biostudies-literatureUnknown22(1)<h4>Background</h4>Mammalian metallothioneins (MTs) are small (6-7 kDa), intracellular, cysteine-rich, metal-binding proteins involved, inter alia, in the homeostasis of zinc and copper, detoxification of heavy metals, antioxidation against reactive oxygen species, and protection against DNA damage. The high cysteine content (~ 30%) in MTs makes them toxic to bacterial cells during protein production, resulting in low yield. To address this issue, we present for the first time a combinatorial approach using the small ubiquitin-like modifier (SUMO) and/or sortase as fusion tags for high-level expression of human MT3 in E. coli and its purification by three different strategies.<h4>Results</h4>Three different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase recoMicrobial cell factoriesCalcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3.PMC103346572019/33/B/ST4/02428Singh AKKrezel AfalseCalcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3.<h4>Background</h4>Mammalian metallothioneins (MTs) are small (6-7 kDa), intracellular, cysteine-rich, metal-binding proteins involved, inter alia, in the homeostasis of zinc and copper, detoxification of heavy metals, antioxidation against reactive oxygen species, and protection against DNA damage. The high cysteine content (~ 30%) in MTs makes them toxic to bacterial cells during protein production, resulting in low yield. To address this issue, we present for the first time a combinatorial approach using the small ubiquitin-like modifier (SUMO) and/or sortase as fusion tags for high-level expression of human MT3 in E. coli and its purification by three different strategies.<h4>Results</h4>Three different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase reco2023-01-01T00:00:00Z2023 Jul2025-04-22T21:11:45.115Z2025-04-22T21:11:45.115ZS-EPMC103346573743413410.1186/s12934-023-02134-x