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Isolation and purification of expression product of Populus euphratica CPD (PeCPD) in Escherichia coli: A comparison of two different methods

Isolation and purification of expression product of Populus euphratica CPD (PeCPD) in Escherichia... An effective procedure for purifying the expression product of Populus euphratica constitutive photomorphogenesis and dwarf (PeCPD) gene in Escherichia coli had been developed. The product, which is a cytochrome P450 monooxygenase, participating in plant brassinosteroid (BR) biosynthesis was deduced. This method is based on the combination of differential centrifugation (DC) with preparative SDS-PAGE and below is named as DC-PSDS-PAGE. The target protein obtained by this method was apparently better in purity and yield than that obtained by Ni2+ chelate affinity chromatography (Ni2+-CAC). The target protein purity and yield was 95% and 37.897 mg/L of bacterial culture, respectively, in DC-PSDS-PAGE vs. 30% and 0.816 mg/L of bacterial culture in Ni2+-CAC. To the best of our knowledge, this procedure is reported for the first time. The reasons for selecting this method and its advantages are discussed in the paper. The purified protein can be used for raising antibody, which is necessary for in situ localization, and this will be a very important part of our subsequent experiments in studying the function of the target protein. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Russian Journal of Plant Physiology Springer Journals

Isolation and purification of expression product of Populus euphratica CPD (PeCPD) in Escherichia coli: A comparison of two different methods

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References (26)

Publisher
Springer Journals
Copyright
Copyright © 2012 by Pleiades Publishing, Ltd.
Subject
Life Sciences; Plant Physiology; Plant Sciences
ISSN
1021-4437
eISSN
1608-3407
DOI
10.1134/S1021443712050068
Publisher site
See Article on Publisher Site

Abstract

An effective procedure for purifying the expression product of Populus euphratica constitutive photomorphogenesis and dwarf (PeCPD) gene in Escherichia coli had been developed. The product, which is a cytochrome P450 monooxygenase, participating in plant brassinosteroid (BR) biosynthesis was deduced. This method is based on the combination of differential centrifugation (DC) with preparative SDS-PAGE and below is named as DC-PSDS-PAGE. The target protein obtained by this method was apparently better in purity and yield than that obtained by Ni2+ chelate affinity chromatography (Ni2+-CAC). The target protein purity and yield was 95% and 37.897 mg/L of bacterial culture, respectively, in DC-PSDS-PAGE vs. 30% and 0.816 mg/L of bacterial culture in Ni2+-CAC. To the best of our knowledge, this procedure is reported for the first time. The reasons for selecting this method and its advantages are discussed in the paper. The purified protein can be used for raising antibody, which is necessary for in situ localization, and this will be a very important part of our subsequent experiments in studying the function of the target protein.

Journal

Russian Journal of Plant PhysiologySpringer Journals

Published: Aug 16, 2012

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