Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3

Abstract

Background: 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.

Results: Three different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase recognition motif (LPETG) as removable fusion tags for high-level expression and purification of human MT3 from the bacterial system. In the first strategy, SUMOylated MT3 was expressed and purified using Ulp1-mediated cleavage. In the second strategy, SUMOylated MT3 with a sortase recognition motif at the N-terminus of MT3 was expressed and purified using sortase-mediated cleavage. In the final strategy, the fusion protein His₆-SUMO-eSrtA-LPETG-MT3 was expressed and purified by one-step sortase-mediated inducible on-bead autocleavage. Using these three strategies the apo-MT3 was purified in a yield of 11.5, 11, and 10.8 mg/L, respectively, which is the highest yield achieved for MT expression and purification to date. No effect of MT3 on Ni²⁺-containing resin was observed.

Conclusion: The SUMO/sortase-based strategy used as the production system for MT3 resulted in a very high expression level and protein production yield. The apo-MT3 purified by this strategy contained an additional glycine residue and had similar metal binding properties as WT-MT3. This SUMO-sortase fusion system is a simple, robust, and inexpensive one-step purification approach for various MTs as well as other toxic proteins with very high yield via immobilized metal affinity chromatography (IMAC).

Keywords: IMAC; Metallothionein; Plasmid generation; Protein purification; Recombinant expression; SUMO; Sortase; Ulp1.

Fig. 1

Schematic representation of sortase-mediated purification of SUMOylated human MT3. Purified SUMO-LPETG-MT3 fusion protein was incubated with eSrtA for 4 h at 37 °C in the presence of 5 mM CaCl₂ for sortase-mediated cleavage of MT3 from the fusion protein. The cleaved G-MT3 was then purified from the reaction mixture using Ni²⁺-IMAC purification

Fig. 2

Schematic representation of Ca²⁺-inducible on-bead autocleavage purification of G-MT3 from SUMOylated human MT3. The fusion protein His6-SUMO-eSrtA-LPETG-MT3 was immobilized/bound on Ni²⁺-NTA beads. On-bead sortase-mediated autocleavage was induced by incubating the beads in 5 mM CaCl₂ for 4 h at 37 °C. Purified G-MT3 was collected in the flow-through

Fig. 3

Plasmid map of the constructed expression vectors encoding fusion proteins SUMO-LPETG-MT3 and sSrtA-MT3

Fig. 4

Purification of fusion proteins. A SDS-PAGE purification profile of SUMO-MT3 and SUMO-LPETG-MT3. Lane 1 = lysate, Lane 2 = supernatant, Lane 3 = flow-through, Lane 4 = wash, Lane 5 = first elution, Lane 6 = second elution, Lane 7 = protein ladder, Lane 8 = second elution, Lane 9 = first elution, Lane 10 = wash, Lane 11 = flow-through, Lane 12 = supernatant, Lane 13 = lysate. B SDS-PAGE purification profile of eSrtA-MT3. Lane 1 = lysate, Lane 2 = supernatant, Lane 3 = flow-through, Lane 4 = wash, Lane 5 = first elution, Lane 6 = second elution, Lane 7 = protein ladder, Lane 8 = protein ladder Bio-Rad. C SDS-PAGE profile of purified fusion proteins. Lane 1 = SUMO-MT3, Lane 2 = SUMO-LPETG-MT3, Lane 3 = protein ladder, Lane 4 = eSrtA-MT3. D SDS-PAGE profile of eSrtA mediated cleavage of SUMO-LPETG-MT3. Lane 1 = fusion protein SUMO-LPETG-MT3, Lane 2 = eSrtA cleaved MT3, Lane 3 = SUMO eluted from beads after cleavage, Lane 4 = proteins from beads, Lane 5 = fusion protein in eSrtA buffer, Lane 6 = empty, and lane 7 = protein ladder Bio-Rad. E Comparison of total protein yield (mg/L) obtained using various purification systems. MT3-WT stands for protein obtained from the IMPACT system

Fig. 5

UV–vis monitored Cd²⁺ titration of (A) 1 µM metal-free G-MT3, GS-MT3, and MT3-WT in 50 mM borate pH 7.4, 100 mM NaClO₄, 100 µM TCEP. Panel (B) indicates saturation isotherms recorded at 240 nm for CdSO₄ titrations. Red vertical lines indicate metal-to-protein ratios where absorbance reaches saturation with Cd²⁺