Isolation of intact FNR protein (Mr 30,000) of Escherichia coli.
FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29,000, which lacks nine N-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30,000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr 29,000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30,000. The N-terminal sequence of FNR (Mr 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys-X5-Cys(29).
Trageser
M
M
Institut für Mikrobiologie, J.W. Goethe-Universität, Frankfurt, FRG.
Spiro
S
S
Duchêne
A
A
Kojro
E
E
Fahrenholz
F
F
Guest
J R
JR
Unden
G
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D004926:Escherichia coli / Q000032:analysis* / Q000235:genetics; D029968:Escherichia coli Proteins; D005784:Gene Amplification; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011480:Protease Inhibitors / Q000494:pharmacology; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification*; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000302:isolation & purification*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D011480:Protease Inhibitors; D012097:Repressor Proteins; D014157:Transcription Factors