CAJ | 학술논문

真核翻译起始因子4A(eukaryotic initiation factor 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(Plasmodium cynomolgi)的cDNA文库,克隆了一个eIF-4A同源蛋白的完整cDNA序列,命名为CH1 F.CHl F全长1753 bp,包含一个1197 bp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-28a(+),在大肠杆菌DH5α中表达,产生的融合蛋白大小在45 kD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.
진핵번역기시인자4A(eukaryotic initiation factor 4A,eIF-4A)시DEAD합단백가족적ATP의뢰성적RNA해선매류중적일개원형성원.타재진핵세포적단백질합성적기시과정중기착관건성작용.통과PCR확증화방사탐침잡교상결합적방법사선식해후학원충(Plasmodium cynomolgi)적cDNA문고,극륭료일개eIF-4A동원단백적완정cDNA서렬,명명위CH1 F.CHl F전장1753 bp,포함일개1197 bp적완정열독광,추측편마일개유398개안기산조성적단백.대CH1F적단백서렬용BlastP진행수색화분석,제시타응해시DEAD합가족적일개eIF-4A동원단백;용DNAStar장기여허다전형적DEAD합단백서렬진행비대분석,결과현시:비기기타적DEAD합단백,타여eIF-4A혹eIF-4A적동원단백구유경고적동원성화경다서렬상적상사결구역.장포함완정열독광적편단아극륭진표체재체pET-28a(+),재대장간균DH5α중표체,산생적융합단백대소재45 kD좌우.대해융합단백진행순화、중신절첩화초보감정.ATP매활성검측현시,해융합단백지유흔저적ATP매활성,이차타적ATP매활성사호불의뢰우핵산저물.대저일검측결과급출3충가능적원인.저일검측결과여근거서렬분석득도적추론--CH1F단백가능시일개eIF-4A병불모순.
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.