Background and overview[1]
In vivo, NTPs/dNTPs participate in the biosynthesis of nucleic acids to generate pyrophosphate as a by-product. The pyrophosphate present in the body will be decomposed under the catalysis of pyrophosphatase, reducing the possible negative effects caused by the accumulation of pyrophosphate. PCR uses thermophilic enzymes to simulate the in vitro reaction of nucleic acid replication in vivo. With the inclusion of dNTPs, the accumulation of pyrophosphate will also reduce the efficiency of PCR amplification. The accumulation of pyrophosphate will cause the pH of the reaction mixture to decrease and affect the stability of the fluorescent dye.
In order to remove the inhibition of PCR by these pyrophosphates, inorganic pyrophosphatase is added to the PCR reaction to hydrolyze pyrophosphate (PPi) into orthophosphate (Pi). This reaction will increase the pH of the reaction mixture and make the fluorescent dye more stable. In order to adapt to the high temperature of PCR, this pyrophosphatase must be able to withstand high temperatures of 95°C. The PPase gene of hyperthermophilic archaea (Pyrococcushorikoshii) was recombinantly expressed. The optimal temperature of this enzyme is 88°C and the optimal pH is 10.3, which is not suitable for PCR. The PPase gene of Thermusthermophiles HB27 strain was recombinantly expressed. Therefore, based on the PPase gene of Thermus thermophilus, a modified thermostable inorganic pyrophosphatase was constructed. This enzyme can be applied to ultra-sensitive PCR amplification that requires more cycles and reaction time, increasing the amplification of the reaction. Increase efficiency. Thermostable Inorganic Pyrophosphatase Features: 1) Enhance DNA replication 2) When heated at 100°C for 4 hours, the enzyme still has 100% activity. ThermostableInorganic Pyrophosphatasecomes from a recombinant E. coli strain carrying an inorganic pyrophosphatase gene cloned from the extremely thermostable Thermococus litoralis.
Preparation[1]
Thermostable inorganic pyrophosphatase, its sequence is:
MANLKTLPVGEKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYP Hanwha PVC paste resin GDYGFIPSTLAEDGDPLDGLVLSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIADVPEGVKQEIQHFFETYKALEAKKGKWVRVTGWRDRAAALEEVKACIARYGK(SEQIDNO: 1).
Modify the site of the above-mentioned enzyme. The site and method of modification are as follows: Amino acid T at position 6 of the original enzyme sequence is modified into amino acid S; amino acid E at position 11 is changed into amino acid K; amino acid at position 72 L is changed to amino acid I or amino acid V at position 73 is changed to amino acid I; amino acid D at position 124 is changed into amino acid A; amino acid A at position 160 is changed into Q or K. Two thermostable pyrophosphatase enzymes with the following amino acid sequences were obtained:
Inorganic pyrophosphatase 1:
MANLKSLPVGKKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAEDGDPLDGIVLSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALEAKKGKWVRVTGWRDRQAALEEVKACIARYGK (SEQ ID NO: 2).
Inorganic pyrophosphatase 2
MANLKTLPVGEKAPEVVNMVIEVPRGSGNKYEYDPGLGVIKLDRVLPGAQFYPGDYGFIPSTLAuv adhesion promoterEDGDPLDGLILSTYPLLPGVVVEVRVVGLLLMEDEKGGDAKIIGVVAEDQRLDHIQDIAAVPEGVKQEIQHFFETYKALEAKKGKWVRVTGWRDRKAALEEVKACIARYGK (SEQ ID NO: 3).
According to the amino acid sequence of the above-mentioned pyrophosphatase, the corresponding base sequence is artificially synthesized, amplified to construct an expression vector, and the protein is expressed and purified.
Main reference materials
[1] CN201710017276.4 A modified thermostable inorganic pyrophosphatase
