Cloning and expression of plant leghemoglobin cDNA of Lupinus luteus in Escherichia coli and purification of the recombinant protein

The yellow lupin leghemoglobin I gene (lbI) was cloned in the pET-3a vector. It was overexpressed in Escherichia coli BL21(DE3)pLysS cells, under the control of T7 RNA polymerase promoter. The recombinant LbI protein, containing E. coli derived heme, was purified to homogeneity using ion exchange and gel filtration chromatography. The recombinant LbI protein has spectral and immunochemical properties identical to LbI isolated from lupin root nodules. The identity between expressed polypeptide and native LbI protein was also supported by microsequencing analysis of the N-terminus of the purified recombinant protein