摘要
通过计算机辅助分析 ,发现在耐热碱性磷酸酯酶 (简称FD TAP)的N端存在 2 6个氨基酸的信号肽序列。但一般信号肽切点并非是完全专一的 ,所以利用基因工程手段将FD TAP的N端分别缺失 2 4、2 5、2 6和 2 7个氨基酸 ,得到了N端分别缺失 2 4、2 5、2 6和 2 7个氨基酸的克隆子 pTAPND2 4、pTAPND2 5、pTAPND2 6和 pTAPND2 7。考虑到这样的克隆子其翻译起始区所形成的能量较低结构稳定的二级结构可能阻碍基因的表达 ,所以在保证氨基酸序列不变的前提下另外设计了 4个引物 ,得到了二级结构能量大大提高的 4个克隆子pTAPND2 4C、pTAP ND2 5C、pTAPND2 6C和 pTAPND2 7C。结果表明pTAPND2 4、pTAPND2 5和 pTAPND2 7没有表达 ;pTAPND2 6和pTAPND2 4C的表达量较低 ,pTAPND2 5C、pTAPND2 6C和pTAPND2 7C的表达量较高。通过测定 pTAPND2 5C、pTAPND2 6C和pTAPND2 7C的表达蛋白TAPND2 5、TAPND2 6和TAPND2 7的比活和热稳定性 ,发现无论比活和热稳定性TAPND2 7明显比TAPND2 5和TAPND2 6高且比野生型FD TAP略高。
Through computer associated analysis,a signal peptide sequence of 26 amino acids was found at N terminal of thermostable alkaline phosphatase (FD TAP). For the cleavage site was usually not identical,four clones named pTAPND24,pTAPND25,pTAPND26 and pTAPND27 with deletion of 24,25,26 and 27 amino acids at N terminal of FD TAP were obtained by genetic engineering. Considered their high stable secondary structure formed by region about translational initial codon which thought to hinder the expression of target gene,four other clones named pTAPND24C,pTAPND25C,pTAPND26C and pTAPND27C were obtained. Their amino acids were identical to pTAPND24,pTAPND25,pTAPND26 and pTAPND27 and their deoxynucleosides were changed a little by which their energy of the secondary structure of the region about translational initial codon increased. These eight clones were expressed in E.coli mph44 which its alkaline phophatase gene was deleted and it was found that pTAPND24,pTAPND25 and pTAPND27 have no expression; pTAPND24C and pTAPND26 have little expression and pTAPND25C,pTAPND26C and pTAPND27C have high expression. The proteins TAPND25,TAPND26 and TAPND27 expressed by pTAPND25C,pTAPND26C and pTAPND27C were purified and their specific activity and thermostability were measured. It was found that the specific activity and thermostablility of TAPND27 was higher than TAPND25,TAPND26 and native FD TAP. The result suggests that TAPND27 was more suitable for application on the condition of hight temperature than the native FD TAP,TAPND25 and TAPND26.
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第6期690-694,共5页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助项目!( 3 9870 40 2 )
关键词
耐热碱性磷酸酯酶
信号肽
表达
Thermostable alkaline phosphatase
signal peptide
mRNA secondary structure
