摘要
金属硫蛋白(Metallothioneins,MTs)由结构独立且功能有明显区别的β,α两个结构域组成,神经生长抑制因子Neuronal Growth Inhibitorx Factor,GIF)又名金属硫蛋白-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,而β-结构域为其功能结构域.为深入系统地研究MTs,尤其是GIF及其结构域的结构与功能,我们构建了金属硫蛋白家族内结构域拼接体βGIF-αXIT-1(βⅢ-α1)和βMT1-αGIF(β1-αⅢ ):PCR扩增得各个结构域的 cDNA序列,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,得率约为80 mg蛋白/L菌液.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.紫外吸收图谱和圆二色性图谱显示,结构域拼接体拥有金属硫蛋白家族成员的特征金属巯基簇结构域初步功能实验表明,βⅢ-α也具有抑制神经元生长的功能.
Metallothioneins(MTs) fold into two separate domains: beta - domain and alpha - domain which obviously differ in function. Neuronal growth inhibitory factor (GIF), named as Metallothionein - III ( MT - III), is first characterized to be capable of inhibiting the growth of neuronal cell in nervous system, and beta - domain is its functional domain. To study deep the structure and function of MTs, GIF and their domains; we construct the splicing - domains within metallothioneins family, beta (GIF) - alpha (MT- 1) (beta (III) - alpha (I)) and beta (MT - I) - alpha (GIF) (beta (I) - alpha (III)) cDNAs were amplified by polymerase chain reaction (PCR), inserted into vector pGEX - 4T - 1, expressed in Escherichia coli as carboxyl terminal extension of glutathione - S - transferase ( GST) by IPTG's induction. After the fusion protein had been digested by thrombin on a Glutathione - Sepharose 4B affinity chromatography column, recombinant beta (III) - alpha (I) and beta (I) - alpha (III) were purified by gel fitration on Sephacryl - S100. Eighty mg of the protein can be obtained from every liter medium after fermentation. The results of SDS - PAGE, amino acids composition, molecular mass, the ratio of metal/protein and sulfhydryl group/protein confirm that the purified protein is the desired one. Ultraviolet ( UV) absorption spectroscopy and circular dichroism ( CD) spectroscopy show splicing - domains have the characteristic metal - sulfhydryl group clusters of metahothionein family. beta (III) - alpha (I), similar with GIF, displays the neuronal growth inhibitory activity.
出处
《化学学报》
SCIE
CAS
CSCD
北大核心
2001年第10期1769-1775,共7页
Acta Chimica Sinica
基金
国家科技攻关项目
关键词
金属硫蛋白
神经生长抑制因子
结构域拼接
发酵
结构
功能
metallothioneins
neuronal growth inhibitory factor
domain splicing
fermentation
structure
function
