MOLECULAR-CLONING OF THE COWPEA LEGHEMOGLOBIN-II GENE AND EXPRESSION OF ITS CDNA IN ESCHERICHIA-COLI - PURIFICATION AND CHARACTERIZATION OFTHE RECOMBINANT PROTEIN

Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have clone d and sequenced the gene that encodes for LbII (lbII), the most abunda nt Lb in cowpea nodules, using total DNA as the template for PCR. Prim ers were designed using the sequence of the soybean Ibc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 ami no acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for Lb II into a constitutive expression vector (pEMBL19(+)) and then express ed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (n LbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis us ing anti-soybean Lb(a) antibodies, tryptic and chymotryptic mapping, a nd spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbI I was reversibly oxygenated/deoxygenated, showing that it is a functio nal hemoglobin.