兰州大学机构库 >生命科学学院
P7C3提高烟酰胺磷酸转移酶(NAMPT)酶活性的结构生物学研究
Alternative TitleStructural research into active P7C3 likewise enhanced the activity of the purified Nicotinamide phosphoribosyltransferase (NAMPT) enzyme
赵红霞
Thesis Advisor刘璞
2015-05-20
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name学士
KeywordNAMPT P7C3 共结晶 X-射线衍射
Abstract烟酰胺磷酸核糖转移酶(NAMPT),也称为内脏脂肪素(visfatin)或前B细胞克隆增强因子(PBEF),在烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide,NAD)补救合成通路中起到关键性作用,可以催化烟酰胺转化为烟酰胺单核苷(nicotinamide mononucleotide,NMN),使NMN迅速转化为NAD。P7C3化合物是通过高通量筛选出的具有促进神经元新生功能的小分子化合物,最新研究已经证实P7C3能够提高NAMPT的酶活力。本文将NAMPT基因构建在pET24a质粒上,用BL21进行转化,并用异丙基硫代半乳糖苷(IPTG)在18℃诱导表达NAMPT,经过His镍柱、FPLC分子筛纯化NAMPT,将纯化的NAMPT稀释到6mg/mL与P7C3混合并共结晶,在PH8.5,沉淀剂浓度为25%的条件下长出晶体,进行晶体X-射线衍射,解析晶体结构,研究P7C3如何调控NAMPT蛋白的功能。
Other AbstractNicotinamide phosphoribosyltransferase(NAMPT),also known as pre-B cell colony-enhancing factor(PBER) or visfatin,is a crucial rate-limiting enzyme of Nicotinamide adenine dinucleotide(NAD) biosynthetic pathway.NAMPT synthesizes nicotinamide mononucleotide(NMN) from nicotinamide,and catalytic NMN converted into NAD quickly.P7C3 is a small molecule compound found by High-throughput screening,that fosters the survival of neurons in cell.The recent research indicated active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme.In our research,full-length human NAMPT was subcloned into the pET24a vector and expressed in BL21 at 18℃ with IPTG. The NAMPT was applied to a Ni-NTA SuperFlow column and FPLC.The purified NAMPT diluted to 6 mg / mL and co-crystallized with P7C3.The protein were grown vapor diffusion drops containing 0.1M Sodium phosphate,PH8.5,25% polyethylene glycol 3350 and 0.1M NaCl.Then,we analysis crystal structure of NAMPT with X-ray diffraction,HKL300, CCP4 ,wincoot and PyMol to find how can P7C3 regulate the activity of the purified NAMPT enzyme.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221901
Collection生命科学学院
Recommended Citation
GB/T 7714
赵红霞. P7C3提高烟酰胺磷酸转移酶(NAMPT)酶活性的结构生物学研究[D]. 兰州. 兰州大学,2015.
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