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.
Russian Journal of Plant Physiology – Springer Journals
Published: Aug 16, 2012
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