神经生长抑制因子的功能结构域双体  

Functional Di-domain of Neuronal Growth Inhibitory Factor

作　　者：季清洲[1] 康巧华[1] 周妍娇[1] 任宏伟[1] 茹炳根[1] 

机构地区：[1]北京大学生命科学学院蛋白质工程国家重点实验室,北京100871

出　　处：《生物化学与生物物理学报》2002年第2期248-252,共5页

基　　金：国家"九五"攻关项目 (No .96 C0 2 0 1 0 9)~~

摘　　要：神经生长抑制因子 (neuronalgrowthinhibitoryfactor,GIF)又名金属硫蛋白 III (metallothionein III,MT III) ,是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白质 ,β结构域为其功能结构域。为深入系统地研究GIF及其结构域的结构与功能 ,构建了神经生长抑制因子功能结构域双体 (GIFβ β)。PCR扩增得到N端 β结构域和C端 β结构域的cDNA ,酶切后克隆入原核表达载体pGEX 4T 1,经发酵、诱导表达、亲和层析、凝血酶切和进一步纯化 ,每升菌液约可得重组GIFβ β蛋白 6 0mg。测其电泳行为、氨基酸组成、质谱、金属巯基含量等 ,证明得到了目的蛋白质。圆二色性图谱显示 ,GIFβ β拥有金属硫蛋白家族成员的特征———金属巯基簇结构域。MTT还原法测定神经元抑制活性大小为 :GIF >GIFβ β>Neuronal growth inhibitory factor (GIF), known also as metallothionein-III (MT-III), was the first validated to be capable of inhibiting growth of neuronal cells in nervous system, its β-domain being funct-ional. GIF functional di-domain (GIFβ-β) was constructed to study the structure and function of GIF. N terminal β-domain and C terminal β-domain cDNAs were amplified by PCR, inserted into vector pGEX-4T-1 and expressed in Escherichia coli, as carboxyl terminal extension of glutathione-S-transferase (GST), by IPTG induction. After digestion by thrombin, the fusion protein was isolated by passing through a glutathione-Sepharose 4B affinity chromatography column and was purified by gel fitration on Sephacryl-S100. About 60 mg protein per liter of bacterial cell culture was achieved. The results of SDS-PAGE, amino acid composition, molecular mass, the ratio of metal/protein and sulfhydryl group/protein showed that the purified protein was the GIFβ-β. Circular dichroism (CD) spectroscopy show GIFβ-β has characteristic metal-sulfhydryl clusters of metallothionein family. Inhibitory activities detected by the MTT reduction assay are: GIF > GIFβ-β > GIF β-domain.

关 键 词：神经生长抑制因子 Β结构域 功能结构域双体 表达 功能 

分 类 号：Q51[生物学—生物化学]