Translocation and neuroprotective properties of transactivator-of-transcription protein-transduction domain-neuroglobin fusion protein in primary cultured cortical neurons.
Ngb (neuroglobin) is a newly discovered hexaco-ordinate globin that is expressed in vertebrate brain and peripheral nervous systems. Expression of Ngb increases in response to oxygen deprivation and protects neurons from hypoxia in vitro and in vivo. However, the lack of its transduction ability into cells resulted in limited neuroprotection. To educe its neuroprotection under hypoxia, a cell-permeable Ngb fusion protein was generated. A rat brain Ngb gene was cloned and fused with a gene fragment encoding the nine-amino-acid TAT PTD (transactivator-of-transcription protein-transduction domain; RKKRRQRRR) of HIV-1 in a prokaryotic expression vector to generate a genetic in-frame N-terminal hexahistidine-tagged) TAT PTD-Ngb fusion protein. It was expressed in soluble form in Escherichia coli BL21(DE3)plysS and purified with Ni(2+)-affinity chromatography. The results showed that the purified fusion protein TAT PTD-Ngb can enter into the primary cultured cortical neurons in a dose-dependent manner when added exogenously to the culture media and can be detected in cells within 48 h. The cell viability under hypoxia was increased and apoptosis induced by hypoxia was decreased after TAT PTD-Ngb was transduced into cortical neurons. The results provide a clue for the research of Ngb and suggest that transduction of TAT PTD-Ngb may be one of the ways for the therapy of CNS (central nervous system) diseases, especially cerebrovascular diseases and neurodegenerative diseases.
Zhou
Guo-Yu
GY
Department of Neurology, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, People's Republic of China.
Zhou
Sheng-Nian
SN
Lou
Zhi-Yin
ZY
Zhu
Can-Sheng
CS
Zheng
Xue-Ping
XP
Hu
Xue-Qiang
XQ
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics; D000860:Hypoxia / Q000473:pathology / Q000628:therapy; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D000078787:Neuroglobin; D009474:Neurons / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000502:physiology
D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D005914:Globins