疏水性ELP基因设计与基因库构建  

Design and construction of hydrophobic ELP genes library

作　　者：林衡[1,2] 李军明[2] 张立超[2] 葛高顺[2] 许崇波[2] 胡学军[2] 

机构地区：[1]大连大学生命科学与技术学院,辽宁大连116622 [2]大连大学医学院,辽宁大连116622

出　　处：《微生物学通报》2013年第4期584-592,共9页Microbiology China

基　　金：国家自然科学基金项目(No.31070822);辽宁省教育厅高校科研计划项目(No.L2010015)

摘　　要：【目的】设计合成疏水性类弹性蛋白(Elastin-like polypeptide,ELP)基因,建立ELP基因库。【方法】选择疏水性最强的异亮氨酸(Ile,I)(疏水参数:4.5)取代ELP五肽重复序列单元(缬氨酸-脯氨酸-甘氨酸-客座氨基酸-甘氨酸,VPGXG)中客座残基X。全基因合成一段含编码(VPGIG)10序列,上游含DraⅢ酶切位点、下游含BglⅠ酶切位点的ELP单元。将DraⅢ和BglⅠ设计为一对同尾酶(Isocaudarner),利用这对同尾酶定向克隆(Recursive directional ligation,RDL)一系列不同拷贝数的ELP基因。为鉴定基因库有效性,随机选取库中ELP[I]50基因进行蛋白表达、纯化并测定其相变温度(Inverse temperaturetransition,Tt)。【结果】建立了ELP[I]n(n=10、20、30、40、50、60、70、80、90、100、110、120)基因库。测定ELP[I]50的Tt为24.3°C。【结论】首次单独选用Ile(I)作为ELP蛋白质标签中客座残基氨基酸,增加了ELP中疏水性氨基酸的含量,为进一步筛选出表达量高、Tt低、分子量小的ELP标签奠定基础。[Objective] To design and synthesis encoding hydrophobic ELP genes and construct ELP genes library. [Methods] The highest hydrophobic He（I） amino acid （hydrophobic pa- rameters： 4.5） was chosen to replace the guest residues X of ELP pentapeptide repeat serial units building block （VPGXG）I0 and the encoding block fragment was synthesized, flanking with a Dra tli and Bgl I sites at upstream and downstream respectively. The Dra III and Bgl I were designed as a pair of isocaudamer for recursive directional clone to construct the ELP genes library. To confirm that the ELP library was constructed properly, the ELP[I]50 gene was randomly chosen to be expressed. The Tt of purified ELP[I]50 was detected via turbidity changing at 0D350. [Results] The ELP genes library containing the ELP[I]n （n=10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120） was successfully constructed. The library was confirmed by expression of ELP[I]50 gene, protein purification by inverse transition cycling. The Tt of ELP[I]50 was 24.3℃. [Conclusion] In order to increase the number of hydrophobic amino acids in ELP, the lie（I） was chosen as a guest amino acid, the foundation for further screening ELP tag was builded up to get proper recombinant tag with smaller molecular weight, high levels expression, and low phase transition temperature.

关 键 词：类弹性蛋白 异亮氨酸 同尾酶 定向克隆 

分 类 号：Q78[生物学—分子生物学]