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. 1992 Sep 16;187(2):1091-7.
doi: 10.1016/0006-291x(92)91309-e.

Engineering the CA(2+)-activated photoprotein aequorin with reduced affinity for calcium

J M Kendall  1 , G Sala-Newby, V Ghalaut, R L Dormer, A K Campbell
Affiliations

Engineering the CA(2+)-activated photoprotein aequorin with reduced affinity for calcium

J M Kendall et al. Biochem Biophys Res Commun. .

Abstract

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.

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