Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein

• Published in: Plant physiology (Bethesda) 1997-06, Vol.114 (2), p.493-500
• Main Authors: Arredondo-Peter, R. (University of Nebraska, Lincoln, NE.), Moran, J.F, Sarath, G, Luan, P, Klucas, R.V
• Format: Article
• Language: English
• Subjects: ADN; Agronomy. Soil science and plant productions; Amino Acid Sequence; ARN RIBOSOMAL; ARN RIBOSOMIAL; Base Sequence; Biological and medical sciences; CHEMICAL COMPOSITION; CLONACION; CLONAGE; CLONING; Cloning, Molecular; CODE GENETIQUE; CODIGO GENETICO; COMPLEMENTARY DNA; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; CONTENIDO PROTEICO; DNA; DNA, Plant; Economic plant physiology; ESCHERICHIA COLI; Escherichia coli - genetics; ESPECTROMETRIA; EXPRESION GENICA; EXPRESSION DES GENES; Fabaceae - genetics; Fabaceae - microbiology; Ferric Compounds; Fundamental and applied biological sciences. Psychology; Gels; GENBANK/U33205; GENBANK/U33207; GENE; GENE EXPRESSION; GENE MAPPING; GENE TRANSFER; GENES; Genes, Plant; GENETIC CODE; GENETIC VECTORS; GLYCINE MAX; LBII GENES; LEGAHEMOGLOBINA; LEGHAEMOGLOBIN; Leghemoglobin - genetics; Leghemoglobin - isolation & purification; LEGHEMOGLOBINE; MOLECULAR SEQUENCE DATA; NODOSITE RACINAIRE; Nodules; NUCLEOTIDE SEQUENCE; NUDOSIDADES RADICULARES; Oxidation-Reduction; Parasitism and symbiosis; PCR; Peptide Mapping; Plant physiology and development; Plant Roots - genetics; Plant Roots - microbiology; Plant-Microbe and Plant-Insect Interactions; Plants; Plants, Medicinal; POLYMERASE CHAIN REACTION; PROTEIN CONTENT; PROTEINAS; PROTEINE; PROTEINS; PURIFICACION; PURIFICATION; Recombinant Proteins - isolation & purification; RIBOSOMAL RNA; RNA; RNA, Messenger - genetics; RNA, Plant - genetics; ROOT NODULES; SECUENCIA NUCLEOTIDICA; Sequence Analysis, DNA; SEQUENCE NUCLEOTIDIQUE; Soybeans; SPECTRAL ANALYSIS; SPECTROMETRIE; SPECTROMETRY; Spectrophotometry; Symbiosis; Symbiosis (nodules, symbiotic nitrogen fixation, mycorrhiza...); TENEUR EN PROTEINES; TRANSFERENCIA DE GENES; TRANSFERT DE GENE; VIGNA UNGUICULATA
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.114.2.493

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 (IbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean Ibc gene. The IbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea IbII 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 Lb(a) 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