Heterologous Expression and Characterization of Mimosinase from Leucaena leucocephala
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Heterologous expression of eukaryotic genes in bacterial system is an important method in synthetic biology to characterize proteins. It is a widely used method, which can be sometimes quite challenging, as a number of factors that act along the path of expression of a transgene to mRNA, and mRNA to protein, can potentially affect the expression of a transgene in a heterologous system. Here, we describe a method for successful cloning and expression of mimosinase-encoding gene from Leucaena leucocephala (leucaena) in E. coli as the heterologous host. Mimosinase is an important enzyme especially in the context of metabolic engineering of plant secondary metabolite as it catalyzes the degradation of mimosine, which is a toxic secondary metabolite found in all Leucaena and Mimosa species. We also describe the methods used for characterization of the recombinant mimosinase.
Key wordsMimosine Mimosinase Leucaena 3-Hydroxy-4-pyridone C-N lyase Aminotransferase Secondary metabolite Heterologous expression
We thank James L Brewbaker (College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa) and Edward J. Behrman for generously providing us leucaena seeds and synthetic 3H4P, respectively. This work was supported by the National Science Foundation Award No. CBET 08-27057 and partially by a HATCH grant (HAW00551-H). VSN was supported by IFP fellowship from the Ford Foundation for 3 years.