title: Pichia pastoris Production of Tat-NGB and Its Neuroprotection on Rat Pheochromocytoma Cells.
abstract: Neuroglobin (NGB) is a newly discovered neuroprotector and mainly localized in the neurons and retinal cells of the central and peripheral nervous systems in vertebrates, and its prokaryotic expression protein of which fused with HIV-1 virus-encoded Tat peptide exhibited significant antioxidant and anti-hypoxia. However, no study has documented on the anti-hypoxia of yeast expressed Tat-NGB. To address it, the NGB cDNA fragment with and without Tat tag was designed and conjugated to pPIC9K followed by electroporation, and positive colonies were screened. Subsequently, Tat-NGB-His and His-NGB-His proteins were expressed by inducer methanol and identified by SDS-PAGE, and purified with HisTrap™ FF crude column. After desalting, the transmembrane transduction of Tat-NGB was examined and identified by Western blot, and the anti-hypoxia activity was also examined by CCK-8 kit. Unexpectedly, Tat-NGB-His and His-NGB-His proteins were high yield and secretory expressed in GS115 Pichia pastoris. After purification, the high purified protein was prepared and exhibited a significant transmembrane transduction of Tat-NGB-His (**p < 0.01, compare to control and His-NGB-His). Significantly, Tat-NGB-His could protect hypoxia induced injury of PC12 cells and had an obviously difference when comparing to control and His-NGB-His groups (*p < 0.05, **p < 0.01). The present study first reported the yeast expressed production of Tat-NGB-His and His-NGB-His, and then elucidated the transduction and neuroprotection of Tat-NGB-His on PC12 cell. It not only provided a significant reference for high-yield expression of NGB in yeast expression system, but also provided a significant prevention and treatment of hypoxic and ischemic brain injury.
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, 100850, People's Republic of China.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Ye
authors.affiliation: Department of Biological Engineering, College of Pharmacy, Jilin University, Changchun, 130021, People's Republic of China.
authors.forename: Yangdong
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, 100850, People's Republic of China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, 100850, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, 100850, People's Republic of China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Li
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, 100850, People's Republic of China.
authors.forename: Weiguang
authors.initials: W
authors.lastname: Li
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, 100850, People's Republic of China. zhangcg@bmi.ac.cn.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D005914:Globins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics; D020925:Hypoxia-Ischemia, Brain / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics; D000078787:Neuroglobin; D018696:Neuroprotective Agents / Q000008:administration & dosage; D016716:PC12 Cells; D010843:Pichia; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D018696:Neuroprotective Agents; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D005914:Globins
keywords: High yield; Neuroglobin; Neuroprotection; Tat peptide; Transduction