title: Stabilisation of recombinant aequorin by polyols: activity, thermostability and limited proteolysis.
abstract: The photoprotein aequorin is a calcium-dependent bioluminescent enzyme which is most widely used in biotechnology processes, but this protein is susceptible to aggregation and proteolysis degradation. Various additives such as polyols are known to enhance the stability of proteins and protect them in native folded and functional state. In this work, for study of aequorin stability, the histidine-tagged apoaequorin was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. Kinetics of light emission of purified aequorin upon addition of Ca(2+) showed a linear dependency on aequorin concentration. Furthermore, the effect of some stabilisers, such as glycerol, glucose, lactose, terehalose, sucrose and sorbitol on thermostability of recombinant aequorin was measured. Results indicate that the recombinant aequorin is very stable in phosphate buffer including 30 mM sorbitol, since after heat shock of 30 min at different temperatures, a slight decrease in activity was observed. However, flexibility and exposure of tryptophan residues of aequorin to the solvent, in the presence and absence of stabilisers, with respect to fluorescence quenching by acrylamide, indicated identical characterisation. In addition, according to limited proteolysis of aequorin demonstrating that this enzyme is sensitive to proteases as in the presence of 2 ng/ml of protease, aequorin was completely digested. In conclusion, sorbitol increases stability of aequorin with high photoactivity and not effect for flexibility and limited proteolysis of this photoprotein.
authors.affiliation: Department of Genetic Engineering, Science and Biotechnology Research Center, Mallek-Ashtar University of Technology, Tehran, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Zeinoddini
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
authors.forename: Ali Reza
authors.initials: AR
authors.lastname: Saeedinia
authors.forename: Seyed-Mortaza
authors.initials: SM
authors.lastname: Robatjazi
mesh_terms: D000331:Aequorin / Q000737:chemistry*; D001059:Apoproteins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D055550:Protein Stability; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry; D013012:Sorbitol / Q000737:chemistry; D013696:Temperature; D014199:Trehalose / Q000737:chemistry
chemical_list: D001059:Apoproteins; D008164:Luminescent Proteins; D011108:Polymers; D011994:Recombinant Proteins; C060014:apoaequorin; C024617:polyol; D013012:Sorbitol; D000331:Aequorin; D014199:Trehalose; D010447:Peptide Hydrolases; D005990:Glycerol; D002118:Calcium