J Integr Plant Biol. ›› 2003, Vol. 45 ›› Issue (7): 858-863.

• Research Articles • Previous Articles     Next Articles

Cloning and Expression of Curcin, a Ribosome-Inactivating Protein from the Seeds of Jatropha curcas

LIN Juan, CHEN Yu, XU Ying, YAN Fang, TANG Lin, CHEN Fang   

Abstract:

Curcin, a ribosome-inactivating protein with a molecular weight of about 28.2 kD, which strongly inhibits the protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about (0.19±0.01) nmol/L, was purified from the seeds of Jatropha curcas L. The protein has the activity of rRNA N-glycosidase. Degenerate primers were designed based on the N-terminal partial sequence from purified curcin. The full-length curcin cDNA by RT-PCR and 5''-RACE was cloned. The deduced amino acids sequence indicates that a preprotein with 293 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids sequence shares homology of 33% and 57% to those of typeⅠribosome-inactivating proteins (RIPs) and A chain of typeⅡRIPs, respectively. The sequence encoding mature curcin was integrated into the pQE-30 vector for expression in Escherichia coil strain M15 (pREP4). The purified recombinant curcin was able to inhibit protein synthesis in rabbit reticulocyte lysate system.

麻疯树核糖体失活蛋白基因的克隆和表达
林 娟 陈 钰 徐 莺 颜 钫 唐 琳 陈 放*
(四川大学生命科学学院,成都 610064)

摘要: 麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,
它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白。从麻疯树种子中分离得到一种分子量为28.2 kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC50 为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性。依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5'-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列。该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽。推测的多肽序列与测定的蛋白质N端序列相同,与多种已发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性。将curcin的编码区与表达载体pQE-30 相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达。将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力。
关键词: 麻疯树;麻疯树核糖体失活蛋白;R NA N -糖苷酶;基因克隆;原核表达

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