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).

Seven oxidative stress-related genes predict the prognosis of hepatocellular carcinoma

“…The study showed that liver cancer patients with lower levels of NPM1 have a better prognosis [ 27 ]. MT3 protects the body from DNA damage, and H2AX is a biomarker of DNA damage [ 28 , 29 ]. As one of the major REDOX regulators, TXNRD1 is associated with tumor aggressiveness and poor prognosis [ 30 ].…”

Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system

“…The complicated IB purification and the recombinant protein refolding are hurdles to large (industrial) scale production. In this study, a simple and rapid recombinant protein production and purification method based on a bacterial surface display system [ 7 ] with calcium-dependent self-cleavage strategy [ 8 , 9 ] was attuned with modification for production of soluble PEN-HuscFv34 super antibody.…”

“…The Lpp′-OmpA′ is a chimeric protein consisting of a signal sequence and the first 9 N -terminal amino acids of the E. coli membrane-anchored lipoprotein (Lpp) which is fused to a transmembrane domain (amino acid residues 46–159) of OmpA (OmpA′) [ 7 ]. This chimeric protein has been used extensively for surface display of recombinant proteins in Gram-negative bacteria [ 7 , 9 – 15 ]. Usually, one cell of wildtype E. coli (with intact OmpA gene) can express as many as 100,000 OmpA molecules on the surface [ 7 ].…”