Affordable Access

deepdyve-link
Publisher Website

Stabilisation of recombinant aequorin by polyols: activity, thermostability and limited proteolysis.

Authors
  • Zeinoddini, Mehdi
  • Khajeh, Khosro
  • Hosseinkhani, Saman
  • Saeedinia, Ali Reza
  • Robatjazi, Seyed-Mortaza
Type
Published Article
Journal
Applied Biochemistry and Biotechnology
Publisher
Springer-Verlag
Publication Date
May 01, 2013
Volume
170
Issue
2
Pages
273–280
Identifiers
DOI: 10.1007/s12010-013-0096-3
PMID: 23504568
Source
Medline
License
Unknown

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.

Report this publication

Statistics

Seen <100 times