信号肽去除的耐热碱性磷酸酯酶FD-TAP在大肠杆菌中的亚克隆和高表达

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２６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２４、２５、２６和２７个氨基酸，得到了Ｎ端分别缺失２４、２５、２６和２７个氨基酸的克隆子ｐＴＡＰＮＤ２４、ｐＴＡＰＮＤ２５、ｐＴＡＰＮＤ２６和ｐＴＡＰＮＤ２７。考虑到这样的无隆子其翻译起始区所形成的能量较低结构稳定的二组

Subcloning and High Expression of Signal peptide Deleted Thermostable Alkaline Phosphatase (FD-TAP)

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.