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| Expression, purification and identification of recombinant human neuroglobin |
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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 signal transduction for neuroprotection of hypoxic and ischemic insults as some researches had been described. However, the underlying mechanism remained unknown. Obviously, it would be played an important role for preparation of neuroglobin in studying its function. 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 identification by mass spectrometry and N-terminal amino acid sequencing analysis. In conclusion, the neuroglobin was not only expressed in soluble form with high-efficiency in E. coli, but also could be easily purified with two steps. The successful expression and purification of the recombinant human neuroglobin will advance the study of neuroglobin either for the neuroprotective function or deciphering the underlying mechanism of hypoxic/ischemic insults.
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Received: 29 April 2009
Published: 10 May 2010
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