Purification of recombinant human apometallothionein-3 and reconstitution with zinc.
Metallothioneins (MT) are small cysteine-rich proteins, expressed in many life forms. They are involved primarily in the metabolism of zinc and copper, and in metal detoxification processes. Metallothionein-3 is a mammalian brain-specific MT, which is down-regulated in Alzheimer's disease brains. In this report, we describe a new procedure for purification of recombinant human apo-MT-3 by three steps, size exclusion at neutral pH, followed by cation-exchange and reverse-phase HPLC, both at low pH. Purified apo-MT-3 was reconstituted with seven Zn(2+) ions and reconstitution products were analyzed with electrospray ionization mass spectrometry. The mass spectrum of reconstituted ZnMT-3 was identical with that of native ZnMT-3 isolated by size exclusion chromatography proving the efficiency of the reconstitution process. It showed that ZnMT-3 exists in solution as a dynamic mixture of several metalloforms, where the main metalloform is Zn(7)MT-3 and minor forms are Zn(6)MT-3 and Zn(8)MT-3.
Eriste
Elo
E
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, Sweden. Elo.Eriste@mbb.ki.se
Kruusel
Keiu
K
Palumaa
Peep
P
Jörnvall
Hans
H
Sillard
Rannar
R
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D001059:Apoproteins; D000090023:Metallothionein 3; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D015032:Zinc