栝楼籽核糖体失活蛋白的纯化、性质及晶体生长研究

Purification,Characterization and Crystal Growth of RibosomeInactivating Protein From the Seeds of Trichosanthes kirilowiiWT〗

栝楼（Ｔｒｉｃｈｏｓａｎｔｈｅｓｋｉｒｉｌｏｗｉ）籽经粉碎抽提、硫酸铵沉淀、阳离子交换及凝胶过滤柱层析等步骤，得到一种单链核糖体失活蛋白－Ｔｒｉｃｈｏｋｉｒｉｎ（ＴＣＫ）．ＳＤＳ－ＰＡＧＥ和ＩＥＦ显示为单一条带，其分子量为２９ｋＤ，ｐＩ≥９．３，含糖量约为１．７５％．该蛋白对兔网织红细胞裂解液系统的蛋白质合成具较强的抑制活性，ＩＣ５０为６．７×１０－１０ｍｏｌ／Ｌ．改进了纯化方法，提高了产率，并培养出晶体．

Ribosome inactivating proteins (RIPs) are a group of toxin with RNA Nglycosidase.It hydrolyzes the Nglycoside bond at position A4324 in the 28S rRNA of the eukaryotic,releasing an adenine and resulting in the inactivating of ribosome.Trichokirin(TCK) is a RIP of single polypeptide chain.A new method for largescale preparation of TCK from the seeds of Trichosanthe kirilowii was established.TCK was isolated and purified to homogeneity by precipitation of (NH4)2SO4,and CMSepharose FF and Sephadex G50 column chromatography.TCK is a glycoprotein containing 175% sugar.The molecular weight of TCK is about 29 000 and pI value higher than 93.Inhibition to protein synthesis was primarily determined by the cell free system of reticulocyte lysate which displayed the IC50 was 67×10-10 mol/L.After screening a large number of conditions for growing,its protein crystal was obtained.