人芳香基硫酸酯酶(HSulf-1)基因的原核表达及酶活初步研究

Prokaryotic Expression and Enzyme Activity Measurement of the Human Arylsulfatase (HSulf-1)

人芳香基硫酸酯酶(HSulf-1)是一类分泌型蛋白,能在中性条件下改变硫酸肝素蛋白聚糖(HSPGs)的硫酸化状态,从而影响多种信号分子与其相应受体的结合,进而影响细胞信号通路。鉴于HSulf-1具有重要的潜在应用价值,为获得大量纯化蛋白以探索其功能与应用,文章将HSulf-1功能域基因片段与pGEX-6p-1表达载体连接构建成重组质粒,在原核表达系统BL21中表达、分离纯化了HSulf-1的功能域片段,并以4-Mus为底物用荧光光度法进行了酶活性的测定。结果表明,原核表达能得到电泳纯级的HSulf-1纯化蛋白,但其具有酶活性的条件需要进一步探索。

The Human arylsulfatase（HSulf-1）,which can alter the sulfation state of Heparan Sulfate Proteoglycans（HSPGs） in the neutral condition,is a secreted protein.The shift of sulfation state of Heparan Sulfate Proteoglycans has the affection on the combination of the signaling molecule and its receptor thereby has regulated the signaling pathway.Given the potential values of HSulf-1＇s application,in order to gain sufficient protein to develop its function and application research,the functional fragment of HSulf-1 is constructed into expression vector,pGEX-6p-1 to form a recombinant plasmid.Expression,isolation,and purification of the fragment of functional domain of Hsulf-1 in the prokaryotic expression system are performed.Then,the enzyme activity of prokaryotic expression product is measured by the method of fluorospectrophotometry using the 4-Mus as substrate.The data suggest that the purified protein can be obtained through the prokaryotic expression system but have no enzyme activity.