Horseradish esterases: detection, purification and identification
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Our goal is to characterize esterases from horseradish tissues and assign their physiological roles. In the present study we focused on isolation, purification and identification of esterases from different horseradish tissues: plantlets and two tumor tissue lines. Horizontal IEF system enabled separation of six esterase isoforms with quite different pI values as well as with pronounced differences in expression levels among analyzed tissues. Esterases were extracted, fractionated by means of cation exchange chromatography, and analyzed by planar gel electrophoresis (SDS–PAGE) and isoelectrical focusing (IEF), UV/Vis spectroscopy, MALDI mass spectrometry (MS) and MALDI-MS/MS. Several chromatographic strategies were applied for esterase purification and characterization. Two subsequent cation exchange chromatographic steps based on SP-Sepharose FF material, followed by in-solution digestion combined with MALDI-MS and MS/MS proved to be the best strategy for identification of two esterase proteins, namely Pectinesterase/pectinesterase inhibitor 18 and GDSL esterase/lipase ESM1.
KeywordsArmoracia lapathifolia Protein identification Protein purification Electrophoresis Mass spectrometry Esterase
This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia, grant No. 098-1191344-2943 and 119-1191196-1200, and Bilateral Cooperation Grant Croatia-Austria (1/2010 to ILA and MMD).
ILA performed purification of esterases, spectroscopy measurements and isoelectric focusing; participated in mass spectrometry measurements and wrote the manuscript. PPŠ performed plant tissue propagations and protein extractions and participated in writing the manuscript. BB performed electrophoretic analyses and participated in writing the manuscript. MMD participated in designing and performing mass spectrometry measurements, as well as interpretation of the results. GA and BKP participated in planning the experiments, interpretation of the results and revising the manuscript. All authors read and approved the final manuscript.
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