Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif) Protein to Heavy Metals Kamala Boonyodying, Thanakorn Watcharasupat, Waranan Yotpanya, Thawatchai Kitti, Wanna Kawang, Duangkamol Kunthalert and Sutthirat Sitthisak Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand 65000 Abstract A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identiied in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif) recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd 2+ , Co 2+ , Cu 2+ and Zn 2+ . The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu 2+ and Zn 2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu 2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding. E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl 2 and CdCl 2 . This study shows that metal binding domain (CXXC motif) recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation. Keywords: heavy metal binding proteins; bioremediation; heavy metals; binding factors; CXXC motif 1. Introduction The treatment of hazardous heavy metal contami- nation in the environment with biological organisms such as plants and microorganisms or their products is known as heavy metal bioremediation (Bonaventura and Johnson, 1997). Microorganisms have an ability to absorb heavy metals by using heavy metal binding- proteins that can degrade, accumulate, or detoxify heavy metals (Huang et al., 2003; Wiatrowski and Barkay, 2005; Bondarenko et al., 2008). These heavy metal- binding proteins are encoded by the metal regulatory genes and heavy metal transporter genes that function as a heavy metal chaperone, heavy metal transport- ers or enzymes that detoxify heavy metals (Sitthisak et al., 2005; Sitthisak et al., 2007; Al Hasin et al., 2010). Heavy metal binding proteins have been identiied in various organisms with the ability to bind and absorb various types of heavy metals (Kao et al., 2008; Al Hasin et al., 2010; Zheng et al., 2011; Zhu et al., 2011). CXXC motif is a metal binding domain that contains Cys-X-X-Cys motif (X is any amino acid). This motif has been found in heavy metal chaperones, in the thiol disulphideoxidoreductase superfamily, in heavy metal transporters from many bacteria, as well as in yeast and in humans (Lutsenko et al., 1997; Huang et al., 2003; Sitthisak et al., 2007; Agarwal et al., 2010). Recently research in bioremediation has focused on genetically engineered bacteria. A number of metal binding proteins from microorganisms are in use to create engineered bacteria for studying bioremedia- tion (Chen et al., 1997; Sing et al., 2011). However, there are some limitations in bioremediation systems as some can remove only one or two heavy metals. Previous work has shown that metal binding proteins, CopA, CopZ and McsA in S. aureus that contains heavy metal binding domain CXXC motifs, are capable of binding various types of heavy metals (Sitthisak et al., 2007; Sitthisak et al., 2012). Understanding the factors affecting the binding of proteins and heavy metals together will provide information of speciicity and selectivity about the heavy metal binding domain CXXC motif, which would then in turn improve analysis about the potential of novel metal binding domain (CXXC motif) recombinant proteins for the purposes of bioremediation. Thus, in this study, an mcsA gene that contains four domains of CXXC motifs from S. aureus was cloned, expressed, and investigated for various environmental factors that are involved in the binding of recombinant proteins and heavy metals. The international journal published by the Thai Society of Higher Education Institutes on Environment Environment Asia Available online at www.tshe.org/EA EnvironmentAsia 5(2) (2012) 70-75