Expression, Purification And The Spectroscopic Characterization Research Of A Particular Plant Metallothionein: Maize MT1

Metallothioneins(MTs) are a family of ubiquitous, low molecular mass,cysteine(Cys)-rich proteins that possess an extraordinary capacity to capture d10 metal ions,found in tissues and organs of nearly all living organisms, including a variety of microorganisms, animal species, plants and human. The biological function of metallothionein is relative to trace elements metabolism, removing toxic heavy metals and scavenging free radicals in organisms. Moreover, it also could influence the growth and development, reproduction, caducity,tumorigenesis, immunity, stress and so on. Compared to a widely researched animal metallothionein, little information is available concerning the possible functions, properties, and structures of plant MTs. In this study, the maize MT1 coding sequence was transferred into the vector, and then the isolation,expression and purification of maize MT1 were conducted by molecular biology methods. It is the first time that maize MT1 has been successfully expressed with high purity in vitro and characterized by gel electrophoresis, protein massspectrometry and so on. The metal ion binding properties and pH stability of the metal-thiolate clusters of maize MT1 were fully characterized by UV absorption spectroscopy. Finally, the effects of Zn(?) chelators, different buffers as well as concentrations of Zn(?) and Cd(?) on the oxidation rate of MT with DTNB have been discussed in detail. The main experiments and conclusions are listed as follows.(1) The maize MT1 gene was cloned and the recombinant protein was expressed as a GST-fusion protein. Maize MT1 was successfully purified and the different factors, such as types of prokaryotic expression vector, Zn2+ion concentration, induction temperature of Isopropyl ?-D-Thiogalactoside, the period of broken bacteria and types of cleavage, on purification and expression of protein has been investigated. The ultimate success was determined that the protein gene was cloned into pGEX-6p-1 vector and expressed in E. coli cells with 50 ?M Zn2+in the medium(5 mL), 1mM IPTG(500 mL) at 37?, followed by Prescission protease cleavage to obtain the desired protein. Maize MT1 was successfully expressed with high purity in vitro for the first time, yielding 3 mg of purified protein per liter of culture medium. This has laid a firm foundation for further studying properties of metallothionein.(2) Optical techniques, such as UV spectroscopy, have generally been employed to probe the fine differences between structures formed for different metals(band energies). The metal binding abilities of maize MT1 were investigated by titrating the protein with incremental amounts of ZnCl2,Cd(NO3)2, or Pb(NO3)2 followed by UV spectroscopy observing the LMCT absorbance bands of the metal-thiolate clusters. Metal ion-binding experiments demonstrate that the binding affinity of maize MT1 follows the order Cd2+>Pb2+> Zn2+. The protein is able to bind two Cd2+ions with an average binding constant of 2.58×106M-1, which is about 5 orders of magnitude lower than that reported for mammalian MTs; no other data for comparison have been reported for plant MTs. Besides, complete release of Zn2+, Cd2+, and Pb2+from metal-thiolate clusters is achieved by acidification below pH ~3. The pH values at which Cd2+, Pb2+, and Zn2+half-dissociate from their corresponding metal-thiolate clusters are ~4.0, ~4.5, and ~5.2, respectively. These values coincide well with the binding affinity of maize MT1 to Cd2+, Pb2+, and Zn2+.The pH stability of the metal-thiolate clusters of maize MT1 is comparable with those of other plant MTs.(3) The reactivity of MT with DTNB was explored by observing the change of characteristic absorption spectra at 412 nm to illustrate the effects of a variety of factors on oxidation rate, including Zn2+chelators, different buffers as well as concentrations of Zn2+and Cd2+. Pseudo-first-order rates were fitted to obtain oxidation rates(kobsd) of MT. Zn2+ions have little effect but cause the inhibition action on the oxidation of MT at high concentrations; while Cd2+ions, as the toxic metal ions, have a stronger affinity for MT and will inhibited the oxidation of MT at low concentrations(2.2 eq). Compared to other chelating agents,EDTA exhibited distinctly trend of accelerating oxidation rate of MT.Commonly used buffers(10mM) increase the reactivity in the following order:HEPES < Tris-HCl < PBS. In short, the thiol reactivity of MT with redox reagents depends on the availability of Zn2+in solution. At low Zn2+concentrations its reactivity of MT is high, whereas at high Zn2+concentrations its reactivity of MT is correspondingly lower.