Molecular cloning, expression, and characterization of a Ca2+-dependent nuclease of Arabidopsis thaliana

Abstract

A gene encoding a Ca2+-dependent nuclease (AtCaN2) consisting of 226 amino acids from Arabidopsis thaliana was isolated. The AtCaN2 gene was expressed in Escherichia coli as a His fusion protein. The His–AtCaN2 fusion protein was purified by affinity chromatography using a Ni–NTA column. Western blot analysis using an anti-His antibody showed that the His–AtCaN2 fusion protein was not degraded. The nuclease activity of the His–AtCaN2 fusion protein was highest at 37°C, required a neutral or weakly-alkaline environment, and was stimulated by Ca2+ and Mg2+, but inhibited by Zn2+ and high concentration of Mn2+.