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

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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.

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Abbreviations

BB:

binding buffer

BR:

brassinosteroids

CBB:

Coomassie brilliant blue

CF:

centrifugal force

DC:

differential centrifugation

EB:

elution buffer

IPTG:

iso-propy-β-D-thiogalactoside

LB:

Luria-Bertani

MAC:

metal-chelated affinity chromatography

Ni2+-CAC:

Ni2+-chelate affinity chromatography

PB:

phosphate buffer

PSDS-PAGE:

preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis

WB:

washing buffer

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Correspondence to X. Y. Wang.

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This text was submitted by the authors in English.

These authors contributed equally to the work.

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Cheng, X.F., Wu, H.J., Zhang, B. et al. Isolation and purification of expression product of Populus euphratica CPD (PeCPD) in Escherichia coli: A comparison of two different methods. Russ J Plant Physiol 59, 684–690 (2012). https://doi.org/10.1134/S1021443712050068

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