Engineering the CA(2+)-activated photoprotein aequorin with reduced affinity for calcium.
Two stage PCR has been used to introduce single amino acid substitutions into the EF hand structures of the Ca(2+)-activated photoprotein aequorin. Transcription of PCR products, followed by cell free translation of the mRNA, allowed characterisation of recombinant proteins in vitro. Substitution of D to A at position 119 produced an active photoprotein with a Ca2+ affinity reduced by a factor of 20 compared to the wild type recombinant aequorin. This recombinant protein will be suitable for measuring Ca2+ inside the endoplasmic reticulum, the mitochondria, endosomes and the outside of live cells.
Kendall
J M
JM
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Sala-Newby
G
G
Ghalaut
V
V
Dormer
R L
RL
Campbell
A K
AK
D000331:Aequorin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D009841:Oligonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000331:Aequorin; D002118:Calcium