Structure of the nifQ gene from Enterobacter agglomerans 333 and its overexpression in Escherichia coli.
The nifQ gene, involved in early stages of iron-molybdenum cofactor (FeMo-co) biosynthesis, was identified downstream of the nifB and nifF genes of Enterobacter agglomerans. This gene was cloned and its nucleotide sequence determined. The amino acid sequence, as deduced from the nucleotide sequence, revealed an accumulation of cysteine amino acid residues at the C-terminal end of the protein. The cysteine cluster showed the following consensus sequence Cys-X4-Cys-X2-Cys-X5-Cys, which is a typical characteristic of metal-binding proteins. Further, the nifQ gene was cloned downstream of strong transcriptional (bacteriophage lambda PLPR) and translational (atpE) signals of the expression vector pCYTEXP1 and expressed as an unfused, soluble protein in Escherichia coli. The molecular mass of 19 kDa, as deduced by SDS-PAGE, is in good agreement with the molecular mass deduced from the nucleotide sequence. The availability of high-level expression clones should facilitate purification of large quantities of the recombinant NifQ protein and elucidation of its properties.
Siddavattam
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D
Lehrstuhl für Genetik, Universität Bayreuth, Germany.
Singh
M
M
Klingmüller
W
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D016384:Consensus Sequence; D017124:Conserved Sequence; D003545:Cysteine / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant / Q000032:analysis; D004754:Enterobacter / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000096:biosynthesis; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D008983:Molybdoferredoxin; C081883:NifQ protein, Pantoea agglomerans; D011994:Recombinant Proteins; D014157:Transcription Factors; D009591:Nitrogenase; D003545:Cysteine