Tandem oligomeric expression of metallothionein from freshwater crab ( Sinopotamon henanense ) in Escherichia coli enhances heavy metal tolerance and bioaccumulation

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal-binding proteins, which play important roles in metal homeostasis and heavy metal detoxification. In our previous study, a novel full length MT cDNA was successfully cloned from the freshwater crab (Sinopotamon henanense). In the present study, tandem repeats of two and three copies of the crab MT gene were integrated by overlap extension PCR (SOE-PCR) and expressed in Escherichia coli. The SUMO fusion expression system was adopted to increase the stability and solubility of the recombinant MT proteins. Furthermore, the metal tolerance and bioaccumulation of E. coli cells expressing oligomeric MTs were determined. Results showed that the recombinant plasmids pET28a-SUMO-2MT and pET28a-SUMO-3MT were successfully constructed with high-fidelity. Oligomeric MTs expression significantly enhanced Cu, Cd and Zn tolerance and accumulation in E.coli. in the order: SUMO-3MT ˃ SUMO-2MT ˃ SUMO-MT ˃ control. Cells harboring pET28a-SUMO -3MT exhibited the highest Cu, Cd and Zn bioaccumulation at 5.8-fold, 3.1-fold and 6.7-fold respectively higher than that of the control cells. Therefore, oligomeric MTs have potential as candidates for heavy metal bioremediation. Our research lays the foundation for the development and application of MTs in the biological treatment of heavy metals. Suggested Reviewers: Wanxi Yang Prof. Ph.D Zhejiang University, China wxyang@spermlab.org Jiang-shiou Hwang Prof. Ph.D National Taiwan Ocean University, Taiwan jshwang@ntou.edu.tw Hans-Uwe Dahms hansudahms@yahoo.com Kadarkarai Murugan Prof. Ph.D Bharathiar University, India kmvvkg@gmail.com Miles Christopher Barnhart Prof. Ph.D Missouri State University, USA christbarnhart@missouristate.edu