[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlappe...[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlapped and served as the template to the other DNA strand in amplification. Then, several rounds of over- lap PCR reaction were conducted to mutate the four cysteines in the HP intein into serines, and introduce a linker sequence containing a tag for product detection and purification. [Result] DNA sequencing analysis proved that the four cysteines in the HP intein were successfully mutated into serines; the PCR precursor fragments were also rejoined into an intact HP gene fragment, without introducing any other unex- pected mutation; the DNA linker of 46 bp was successfully inserted into the de- signed HP gene sequences, without any mutation and base pair mismatch. [Conclu- sion] The HP intein was rapidly reconstructed by overlap-extension PCR in this study, which not only expands the application of overlap PCR in protein engineering, but also provides a simple, efficient and highly accurate tool for the reconstruction and modification of intein.展开更多
基金Supported by the National High Technology Research and Development Program of China(863Program)(2006AA03Z451)Shanghai Basic Research Key Program(10JC1400300)+1 种基金National Natural Science Foundation of China(31070698)the Fundamental Research Funds for the Central Universities(12D10520)~~
文摘[Objective] This study aimed to reconstruct the HP intein by overlap-exten- sion PCR. [Method] Several primers were designed according to the principle of overlap-extension PCR that the repeat sequences were overlapped and served as the template to the other DNA strand in amplification. Then, several rounds of over- lap PCR reaction were conducted to mutate the four cysteines in the HP intein into serines, and introduce a linker sequence containing a tag for product detection and purification. [Result] DNA sequencing analysis proved that the four cysteines in the HP intein were successfully mutated into serines; the PCR precursor fragments were also rejoined into an intact HP gene fragment, without introducing any other unex- pected mutation; the DNA linker of 46 bp was successfully inserted into the de- signed HP gene sequences, without any mutation and base pair mismatch. [Conclu- sion] The HP intein was rapidly reconstructed by overlap-extension PCR in this study, which not only expands the application of overlap PCR in protein engineering, but also provides a simple, efficient and highly accurate tool for the reconstruction and modification of intein.
