食蟹猴疟原虫 (Plasmodium cynomolgi)中一个真核翻译起始因子4A(eIF-4A)同源蛋白的cDNA克隆、表达和ATP酶活性测定(英文)

真核翻译起始因子 4A(eukaryoticinitiationfactor 4A ,eIF 4A)是DEAD盒蛋白家族的ATP依赖性的RNA解旋酶类中的一个原型成员 .它在真核细胞的蛋白质合成的起始过程中起着关键性作用 .通过PCR扩增和放射探针杂交相结合的方法筛选食蟹猴疟原虫 (Plasmodiumcynomolgi)的cDNA文库 ,克隆了一个eIF 4A同源蛋白的完整cDNA序列 ,命名为CH1F .CH1F全长 1 75 3bp ,包含一个1 1 97bp的完整阅读框 ,推测编码一个由 398个氨基酸组成的蛋白 .对CH1F的蛋白序列用BlastP进行搜索和分析 ,提示它应该是DEAD盒家族的一个eIF 4A同源蛋白 ;用DNAStar将其与许多典型的DEAD盒蛋白序列进行比对分析 ,结果显示 :比起其它的DEAD盒蛋白 ,它与eIF 4A或eIF 4A的同源蛋白具有更高的同源性和更多序列上的相似结构域 .将包含完整阅读框的片段亚克隆进表达载体pET 2 8a (+) ,在大肠杆菌DH5α中表达 ,产生的融合蛋白大小在 4 5kD左右 .对该融合蛋白进行纯化、重新折叠和初步鉴定 .ATP酶活性检测显示 ,该融合蛋白只有很低的ATP酶活性 ,而且它的ATP酶活性似乎不依赖于核酸底物 .对这一检测结果给出 3种可能的原因 .这一检测结果与根据序列分析得到的推论———CH1F蛋白可能是一个eIF 4A并不矛盾

cDNA Cloning, Expression and ATPase Assay of a Putative eIF-4A of Plasmodium cynomolgi

Eukaryotic initiation factor 4A (eIF-4A), a prototype of the DEAD-box family of ATP-dependent RNA helicases, plays a pivotal role in the initiation of protein synthesis in eukaryotic systems. CH1 F, the full-length cDNA of a putative eIF-4A, was isolated by screening the cDNA library of Plasmodium cynomolgi,and then expressed in E. coli DHSα. CH1 F is composed of 1753 bp including the ORF of 1197 bp encoding a protein of 398 amino acid residues. Alignment analyses indicated that the deduced protein sequence was much more similar to other eIF-4As than other DEAD-box proteins. The recombinant protein of about 45 kD was purified, refolded and characterized. The ATPase assay showed that ATPase activity of the fusion protein was very low and seemed independent of nucleic acids. Here are three possible explanations for the results,and the results have no contradictions with the assumption that CH1F is an eIF-4A, derived from alignment analyses.