Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein.
Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino 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 LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) 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 using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.
Arredondo-Peter
R
R
Department of Biochemistry, University of Nebraska, Beadle Center, Lincoln 68588-0664, USA. ra@unlinfo.unl.edu
Moran
J F
JF
Sarath
G
G
Luan
P
P
Klucas
R V
RV
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000235:genetics* / Q000382:microbiology; D005290:Ferric Compounds; D017343:Genes, Plant; D007874:Leghemoglobin / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D018517:Plant Roots / Q000235:genetics / Q000382:microbiology; D010946:Plants, Medicinal; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry
D018744:DNA, Plant; D005290:Ferric Compounds; D007874:Leghemoglobin; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; C056771:leghemoglobin II