Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein.
The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ702, CAB was found to be expressed at higher levels than in S. erythraea, with no apparent effects on either growth or sporulation. A more convenient expression system for CAB was obtained by introducing an NdeI site at the initiation codon by using oligonucleotide-directed mutagenesis and placing the gene in the expression vector pT7-7 in Escherichia coli. In this system, CAB was efficiently expressed at levels up to 20 to 30% of total cell protein. When purified to homogeneity from either E. coli or Streptomyces lividans, CAB was found to be identical to the protein previously obtained from S. erythraea.
Swan
D G
DG
Department of Biochemistry, University of Cambridge, United Kingdom.
Cortes
J
J
Hale
R S
RS
Leadlay
P F
PF
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000648:ultrastructure; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*
D002135:Calcium-Binding Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins