The chloroplast small heat shock protein--purification and characterization of pea recombinant protein.
We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using an Escherichia coli expression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography. After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step. The purified recombinant protein had an isoelectric point of 5. 0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in plants after heat-stress treatment. This chloroplast-localized heat shock protein belongs to a special group of small heat shock proteins (sHSPs), which share an evolutionary conserved C-terminal domain with the vertebrate eye lens alpha-crystallin. The crystallins are known from both crystallographic and spectroscopic data to be all-beta proteins. In contrast, this paper presents circular dichroism spectroscopy data which shows that the purified recombinant HSP21 oligomer has a content of more than 30% alpha-helical secondary structure.
Department of Biochemistry, University of Lund, Lund, S-221 00, Sweden.
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Härndahl
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Tufvesson
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Sundby
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002736:Chloroplasts / Q000737:chemistry*; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D018532:Pisum sativum / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000596:Amino Acids; D003459:Crystallins; D006360:Heat-Shock Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins