The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli.
The pseudoazurin gene from Thiosphaera pantotropha has been cloned and sequenced. The deduced amino acid sequence showed that the protein contains an unusually alanine-rich signal peptide, 22 amino acid residues in length, which targets the protein to the periplasm. This pseudoazurin was expressed in large amounts in the periplasm of Escherichia coli when the gene with its native ribosome-binding site was placed downstream of the lac promoter. Removal of a putative hairpin-forming structure upstream of the ribosome-binding site increased the yield of the purified protein to approximately 80 mg/l. The recombinant protein is indistinguishable from that purified from its natural host. A primer extension study indicated that the pseudoazurin structural gene (pazS) is under the control of the Fnr/Nnr regulatory system, but no promoter-binding sequence could be recognized. The amino acid sequence of pseudoazurin from Paracoccus denitrificans is also reported.
Leung
Y C
YC
Oxford Centre for Molecular Sciences, University of Oxford, U.K.
Chan
C
C
Reader
J S
JS
Willis
A C
AC
van Spanning
R J
RJ
Ferguson
S J
SJ
Radford
S E
SE
D000595:Amino Acid Sequence; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010230:Paracoccus / Q000737:chemistry*; D010231:Paracoccus denitrificans / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D017421:Sequence Analysis
D017931:DNA Primers; D011994:Recombinant Proteins; C047163:pseudoazurin; D001400:Azurin