Cloning, expression, and purification of the general stress protein YhbO from Escherichia coli.
We cloned, expressed, and purified the Escherichia coli yhbO gene product, which is an amino acid sequence homolog to the Bacillus subtilis general stress protein 18 (the yfkM gene product), the Pyrococcus furiosus intracellular protease PfpI, and the human Parkinson disease protein DJ-1. The gene coding for YhbO was generated by amplifying the yhbO gene from E. coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21 (DE3) E. coli strain transformed with the YhbO-expression vector, pET-21a-yhbO, accumulates large amounts of a soluble protein with a molecular mass of 20 kDa in SDS-PAGE that matches the expected YhbO molecular weight. YhbO was purified to homogeneity by ion exchange chromatography and hydroxyapatite chromatography, and its identity was confirmed by N-terminal sequencing and mass spectrometry analysis. The native protein exists in monomeric, trimeric, and hexameric forms. We also report a strong sequence homology between YhbO and the general stress protein YfkM (64% identities), which suggests that YhbO is a stress protein, and a strong structural homology between YhbO and the Pyrococcus horikoshii intracellular protease PhpI. We could not, however, detect any proteolytic or peptidolytic activity of YhbO, using classical biochemical substrates.
Abdallah
Jad
J
Stress Molecules, Institut Jacques Monod, Universite Paris 7, France.
Kern
Renee
R
Malki
Abderrahim
A
Eckey
Viola
V
Richarme
Gilbert
G
D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D046912:Multiprotein Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D015513:Oncogene Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D000071617:Protein Deglycase DJ-1; D012689:Sequence Homology, Nucleic Acid
D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; D015513:Oncogene Proteins; C513540:YhbO protein, E coli; C105131:PARK7 protein, human; D000071617:Protein Deglycase DJ-1