ABSTRACT
Neuroglobin(NGB),widely and specially expressed in neurons of various vertebrates,was reported to be a scavenger of reactive oxygen species and/or a stress-responsive sensor for neuroprotection of hypoxic and ischemic insults.However,the underlying mechanism remained unknown.It is important for the functional study of NGB by preparing the protein samples.To address it,the human neuroglobin cDNA fragment was amplified by RT-PCR from human fetus brain and cloned into the prokaryotic expression vector pBV220,and then transformed into E.coli HB101 cells for expression.The expressed protein was purified by gel filtration and anion exchange column followed with SDS-PAGE and Western blot identification,and then desalting by sephadex G-25 medium.The prepared neuroglobin was further identified by mass spectrometry and N-terminal amino acid sequencing analysis.The expressed bacterium,the lysate supernatant and the purified protein samples had visible red color,showing a typical activity of the globin family proteins.In conclusion,the neuroglobin was not only expressed in soluble form with high-efficiency in E.coli,but could also be easily purified with only two steps.The preparation of the NGB proteins will advance the neuroprotective function and mechanism studies of the novel globin.