Cloning, Recombinant Expression and Characterization of Wild Type‐105‐Trp‐Calmodulin of the Green Alga Mougeotia scalaris

The single calmodulin gene (CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the CDNA inserted into the prokaryotic expression vector pGEX‐2T. The recombinant calmodulin protein (CAM) was expressed as a fusion product together with glutathione S‐transferase and isolated on glutathione sepharose. After cleavage and purification, the CaM was characterized by Ca2+‐dependent shift in SDS‐PAGE, by activation of cyclic 3′,5′nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca2+ buffers in the PDE test, affinity to Ca2+ of Mougeotia CaM was found to be diminished fivefold compared to maize or bovine brain CaMs. There was also a 20‐fold increase of half maximal activation (Kact) in the PDE test for Mougeotia CaM relative to maize CaM, while the Kact of maize CaM to that of bovine brain CaM was almost the same. The derived amino acid sequences of CaM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105‐Trp (Mougeotia) → Leu (maize). In Mougeotia CaM the 105‐Trp, including the neighbouring side chains of 92‐Phe and 141‐Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling.