Study on metal binding capacity of the freshwater crab Sinopotamon henanense's recombinant copper specific binding metallothionein expressed in Escherichia coli

Abstract

The copper specific binding metallothionein (CuMT) is a type of cysteine-rich, metal-binding, small protein which plays an important role in Cu²⁺ metabolism in vertebrates. In this study, we investigated the metal tolerance and removing ability of recombinant strains harboring CuMT obtained in vivo from the freshwater crab Sinopotamon henanense (ShCuMT) in order to study its physiological functions and metal binding capacity. We performed a 3D modeling of ShCuMT and created its structural and functional models using the I-TASSER program. The shCumt gene was inserted into a pGEX-4t-1 vector and recombinant soluble ShCuMT was expressed in Escherichia coli. In addition, in order to characterize the tolerance and removing ability of heavy metals in E. coli with ShCuMT expression, the recombinant strains harboring ShCuMT were exposed to various concentrations of Cd²⁺, Cu²⁺ and Zn²⁺, respectively. The results showed that ShCuMT contains transition metal binding sites. In addition, E. coli cells expressing ShCuMT exhibited enhanced metal tolerance and higher removing ability of metal ions than control cells. However, compared with Cd²⁺ and Zn²⁺, E. coli cells expressing ShCuMT have stronger tolerance and higher removing ability of Cu²⁺. In general, ShCuMT contains multiple transition metal binding sites, and it could enhance tolerance and removing ability of metal ions. Therefore, ShCuMT can provide potential candidates for heavy metal bioremediation. This research on the metal binding properties of ShCuMT provides a scientific basis for bioremediation of heavy metal pollution by the recombinant strains.

Keywords: Bioremediation; Copper specific binding metallothionein; Molecular modeling; Sinopotamon henanense.