天花粉蛋白的定点聚乙二醇修饰

Site-Directed Polyethylene Glycol Modification of Trichosanthin

用一种定点修饰天花粉蛋白（ｔｒｉｃｈｏｓａｎｔｈｉｎ，ＴＣＳ）的方法，将聚乙二醇（ＰＥＧ）偶联到预先选定的位点．利用ｎＴＣＳ无半胱氨酸（Ｃｙｓ）残基这一特点，通过定点突变将一个Ｃｙｓ残基引入ＴＣＳ以取代第７位的丝氨酸（Ｓｅｒ）残基．然后，与巯基反应的ＰＥＧ－ｍ ａｌｅｉｍ ｉｄｅ 即可偶联到新引入的Ｃｙｓ 残基上．经纯化得到均一的ＰＥＧ－ＴＣＳ复合物，在ＳＤＳ－ＰＡＧＥ上显示一条区带，表观分子量为３８ ｋＤ．复合物的体外致核糖体失活活性降低了６倍，但其体内引产活性与ｎＴＣＳ相同．定点ＰＥＧ修饰方法为改造ＴＣＳ提供了新途径．

The site directed polyethylene glycol(PEG)modification(PEGylation)had been establishied to couple PEG to a pre selected site on trichosanthin(TCS)which in its natural form had no Cys residue.By site directed mutagenesis,a Cys residue can be introduced to replace the 7th residue Ser and PEG was then coupled to this newly created Cys by PEG maleimide.The PEG TCS conjugate was purified to homogeneity and showed a single band on SDS PAGE with an apparent molecular weight of 38 kD.Its ribosome inactivating activity in vitro was reduced by 6 fold,whereas the abortifacient activity in vivo was comparable to nTCS.The site directed PEGylation has provided a new approach for reconstructing TCS.