Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography.
In this study we describe a novel method for purification of Arabidopsis thaliana dehydrins overproduced in Escherichia coli. The cDNAs corresponding to the four dehydrin genes RAB18, LTI29, LTI30, and COR47 were inserted into a bacterial expression vector under an isopropyl beta-d-thiogalactopyranoside (IPTG) inducible bacterial promoter. After IPTG induction all four proteins accumulated in high amounts. The recombinant proteins were efficiently purified to over 95% purity with a three-step purification scheme: heat fractionation, immobilized metal ion affinity chromatography (IMAC), and ion exchange chromatography. In this study we introduce the novel use of IMAC as an efficient purification method for native dehydrins. Characterization of the purified proteins was done by Edman degradation, mass spectrometry, reverse-phase chromatography, and analytical gel filtration under native and denaturing conditions. Yields of purified proteins were between 2.8 and 12.5 mg per liter of bacterial culture, sufficient for further biochemical studies.
Svensson
J
J
Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala, S-750 07, Sweden. Jan.Svensson@vbiol.slu.se
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Welin
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D017360:Arabidopsis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006358:Hot Temperature; D013058:Mass Spectrometry; D008670:Metals / Q000378:metabolism*; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D008670:Metals; D010940:Plant Proteins; D011994:Recombinant Proteins; C070556:dehydrin proteins, plant