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.