山西大学学报（自然科学版）
山西大學學報（自然科學版）
산서대학학보（자연과학판）
JOURNAL OF SHANXI UNIVERSITY
2012年
2期
369-375
,共7页
八肋游仆虫%肽链释放因子1a%表达%Pull-down分析
八肋遊僕蟲%肽鏈釋放因子1a%錶達%Pull-down分析
팔륵유부충%태련석방인자1a%표체%Pull-down분석
Euplotes octoca rinatus%eRF1a%expression%Pull-down assay
真核生物蛋白质合成终止需要两类肽链释放因子,eRF1和eRF3.研究表明八肋游仆虫的两类肽链释放因子在体内和体外都能形成复合物,且第一类肽链释放因子eRF1a与第二类肽链释放因子eRF3的C端结合.为了确定eRF3在eRF1a上的结合区,本研究以八肋游仆虫第一类肽链释放因子eRF1a基因为模板,用PCR的方法获得了N端分别截短140和206个氨基酸的eRF1a片段,同时在这两个片段的3'端融合了编码6个组氨酸残基的核苷酸序列,将这两个序列分别插入原核表达载体pTWIN1,并构建重组表达质粒pTWIN1-eRF1aC1 his6和pTWIN1-eRF1 aC2his6,转入大肠杆菌BL21( DE3)中获得了可溶性表达,通过一步His60 Ni Superflow柱亲和层析,重组蛋白CBD-intein-eRF1 aC1 his6和CBD-intein-eRF1aC2 his6获得纯化.体外pull down分析显示eRF1aC1和eRF1aC2均能与八肋游仆虫第二类释放因子eRF3相互作用,这表明八肋游仆虫eRF1a的C端是肽链释放因子eRF3的结合区.
真覈生物蛋白質閤成終止需要兩類肽鏈釋放因子,eRF1和eRF3.研究錶明八肋遊僕蟲的兩類肽鏈釋放因子在體內和體外都能形成複閤物,且第一類肽鏈釋放因子eRF1a與第二類肽鏈釋放因子eRF3的C耑結閤.為瞭確定eRF3在eRF1a上的結閤區,本研究以八肋遊僕蟲第一類肽鏈釋放因子eRF1a基因為模闆,用PCR的方法穫得瞭N耑分彆截短140和206箇氨基痠的eRF1a片段,同時在這兩箇片段的3'耑融閤瞭編碼6箇組氨痠殘基的覈苷痠序列,將這兩箇序列分彆插入原覈錶達載體pTWIN1,併構建重組錶達質粒pTWIN1-eRF1aC1 his6和pTWIN1-eRF1 aC2his6,轉入大腸桿菌BL21( DE3)中穫得瞭可溶性錶達,通過一步His60 Ni Superflow柱親和層析,重組蛋白CBD-intein-eRF1 aC1 his6和CBD-intein-eRF1aC2 his6穫得純化.體外pull down分析顯示eRF1aC1和eRF1aC2均能與八肋遊僕蟲第二類釋放因子eRF3相互作用,這錶明八肋遊僕蟲eRF1a的C耑是肽鏈釋放因子eRF3的結閤區.
진핵생물단백질합성종지수요량류태련석방인자,eRF1화eRF3.연구표명팔륵유부충적량류태련석방인자재체내화체외도능형성복합물,차제일류태련석방인자eRF1a여제이류태련석방인자eRF3적C단결합.위료학정eRF3재eRF1a상적결합구,본연구이팔륵유부충제일류태련석방인자eRF1a기인위모판,용PCR적방법획득료N단분별절단140화206개안기산적eRF1a편단,동시재저량개편단적3'단융합료편마6개조안산잔기적핵감산서렬,장저량개서렬분별삽입원핵표체재체pTWIN1,병구건중조표체질립pTWIN1-eRF1aC1 his6화pTWIN1-eRF1 aC2his6,전입대장간균BL21( DE3)중획득료가용성표체,통과일보His60 Ni Superflow주친화층석,중조단백CBD-intein-eRF1 aC1 his6화CBD-intein-eRF1aC2 his6획득순화.체외pull down분석현시eRF1aC1화eRF1aC2균능여팔륵유부충제이류석방인자eRF3상호작용,저표명팔륵유부충eRF1a적C단시태련석방인자eRF3적결합구.
In eukaryotes,translation termination is mediated by two polypeptide chain-release factors,eRF1 and eRF3.In Euplotes octocarinatus,eRF1 and eRF3 forms complex in vivo and in vitro,and the eRF1a binding region on eRF3 are located at the C terminus of eRF3.To identify the eRF3 binding region on eRF1a,the gene-coding regions of two N-terminal truncated eRF1a,eRF1aC1 (aa 141-447) and eRF1aC2 (aa 207-447) were inserted into pTWIN1 vector fused in frame with an DnaB intein gene; meanwhile,a hexahistidine sequence was fused to the C terminus of CBD-intein-eRF1aC1 and CBD-intein-eRF1aC2.The recombinant plasmids pTWIN1-eRF1aC1his6 and pTWIN1eRF1aC2his6 were transformed into the strain E.coli BL21 (DE3).The recombinant CBD-intein-eRF1aC1his6 and CBD-intein-eRF1aC2his6 proteins were expressed and then purified by one-step affinity chromatography using His60 Ni Superflow column.In vitro pull-down analysis showed that the recombinant CBD-intein-eRF1aC1his6 and CBD-intein-eRF1aC2his6 interacted with E.octocarinatus polypepride chain release factor eRF3.This result suggested that C terminus of eRF1a were the binding region of release factor eRF3 in Euplotes octocarinatus.
