Expression, purification, and characterization of metallothionein-A from rainbow trout Laura Vergani, a, * Myriam Grattarola, a Francesco Dondero, b and Aldo Viarengo b a Department of Biophysical M. & O. Sciences and Technologies, Biophysical Division, University of Genova Medical School, Corso Europa 30, Genoa 16132, Italy b Department of Science and Advanced Technology, University of Piemonte Orientale, Corso Borsalino 54, Alessandria 15100, Italy Received 5 August 2002, and in revised form 25 September 2002 Abstract Recombinant metallothionein A (MT-A) from rainbow trout has been successfully produced in milligram quantities in Escherichia coli.cDNAhasbeensubclonedintopGEX-6P.1vector,in-framewithasequenceencodinganN-terminalglutathione- S-transferase (GST) tail. Purification to electrophoretic homogeneity has been obtained by affinity chromatography using GSH– Sepharose. After enzymatic cleavage of GST tail, the MT-A moiety shows a molecular weight, corresponding to the expected one (6630Da). The final yield of the entire expression and purification process was about 5mg of pure metallothionein per liter of bacterialculture.Theeffectsofdifferentreducingandalkylatingagentshavebeenevaluatedattheleveloftheformationofhigher molecular weight aggregates. To investigate the metal-binding ability of the recombinant MT-A, we carried out a spectrophoto- metricaltitrationwithcadmiumions.Finally,wecheckedthemetaldissociationbyrecordingtheUVabsorbanceoftheproteinasa function of the environmental pH. Ó 2002 Elsevier Science (USA). All rights reserved. Metallothioneins (MTs) constitute a superfamily of low-molecular-weight, cysteine-rich proteins, with high affinity for both essential and non-essential metals [1]. MTs are widely expressed in organisms; in eukaryotes, they are responsible for essential metal metabolism and heavy metal detoxification [2,3]. MTs also play an an- tioxidative role: in vitro they display an oxyradical scavengingcapacity,suggestingthattheymayneutralize hydroxyl radicals in vivo [4–7]. As mentioned above, they are thought to function mainly as intracellular distributors and mediators of essential metals, such as zincandcopper[8].Moreover,ithasbeenobservedthat MTs can be oxidized in vitro and the subsequently released zinc may play an important role in gene regulation [9]. Atageneticlevel,ithasbeenobservedthatMTgenes are readily induced by various physiological and toxi- cological stimuli, such as heavy metals, mitogens, cyto- kines, hormones, inflammation and immune response, heat, chemicals and drugs, etc. [10–15]. This evidence hassuggestedadetoxificationandprotectivefunctionof MT overexpression. From a structural point of view, metallothioneins show an unusual amino acid composition that shows lackofaromaticaminoacidsandhistidineandplentyof cysteine residues (up to 30%). The latter are distributed intypicalmotifsequencessuchasCys–Cys,Cys–X–Cys, andCys–XY–CYS(whereXandYstandforanamino acidresidueotherthanCys).Itiswellknownthatthese sulfydryl residues are able to bind copper, zinc, and cadmium with high affinity, through the formation of metal-thiolate clusters [2]. The unusual features of me- tallothioneins provoked the increasing interest of the scientific community in the investigation of the struc- ture–function relationship of these proteins. All examined vertebrates contain two, or more, dis- tinct MT isoforms (MT-I–IV). The occurrence of mul- tiple forms of MTs in a single species suggests specific biological functions of the various isoforms. Human MT-IIa, but not MT-I, in fact, is widely expressed in most cells types also in the absence of inducers [16]. Conversely, human MT-III and MT-IV are expressed at basal levels, in differentiating stratified squamous Protein Expression and Purification 27 (2003) 338–345 www.elsevier.com/locate/yprep * Corresponding author. Fax: +39-10-3538346. E-mail address: vergani@ibf.unige.it (L. Vergani). 1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1046-5928(02)00631-9