Study On Site-directed Mutagenesis Of Taq DNA Polymerase And Its Fidelity

In vitro directed enzyme evolution is a new technology which develops in recent years.It has widely succeeded used in the in vitro directed improvement of enzyme.Today,Easy wrong PCR and DNA Shuffling which based on homologous reorganization are the main technologies used in this domain,however,the core of these technologies are in vitro mutagenesis which based on PCR process and eventually realizes the molecular multiplicity,but the procedure complexity causes the limited application.In order to make this technology to be simple and practical,attempts to reduce the fidelity of Taq DNA polymerase and further make it more adaptable for the PCR procedure of directed genetic mutagenesis.According to the structure and the function information of Taq DNA polymerase and other research foundation of this enzyme,this research carried on site-directed mutagenesis and the fidelity research to Taq DNA polymerase,and then by using the low fidelity Taq DNA polymerase constructed before,proceeded the in vitro evolution of cellulase which is of widely concern at present, and obtained results as following:1.Through long primer amplification strategy to turn codon ACC to codon CGC at T664 spot of Taq DNA polymerase,causes the transit of Threonine to Arginine,and obtains Taq T664R mutant,And take GFP as the template,analyzed the fidelity of this mutant through PCR product clone and the sequence determinationThe result indicated that,the total mutation rate of Taq-V661H is 0.122%,mutation frequency enhancement 6 times compared to the wild type.Interesting is,95%base mutation occurs in AT base.And among them,mutant frequency of A to G account for 48%of total mutant frequency.This phenomenon indicated that the V661H spot is not only involved with reducing the fidelity of Taq DNA polymerase,but also related to the selectivity of base mutant.This new discovery has provided new information for the structure and the function relations of Taq DNA polymerase,meanwhile,laid the foundation for its further applications.2.Turn codon CAA to codon CGC at 666 spot through site-directed mutagenesis,causes conversion of glutamine to arginine,and obtains Taq-N666R mutant.Similarly,analyzed the fidelity of TAQ-N666R by taking GFP as the template,the result indicated that total mutant frequency is 0.042%, and discovered the mutant frequency of this mutant has obvious difference to different base,among them,mutant frequency of T to C account for 42%of total mutant frequency.This mutant characteristic provide new clue for further research of the base mutant selectivity of Taq DNA polymerase.3.Turn codon ACC to codon CGC at 664 spot of Taq DNA polymerase through site-directed mutagenesis,caused threonine to become arginine and has obtained Taq-T664R mutant.Used this mutant enzyme to carry on PCR amplification,analyzed the influence of Taq-T664R to the mutant frequency of GFP base.The result indicated that,the total rate of base mutation is 0.99%,the mutant frequency increased 99 times compared to wild type,and discovered the ratio of A and T transform for other base account for 95%,and the ratio of T transform for C and A transform for G account for 18%and 42%respectively.In the protein level,the rate of amino acid mutation is 2.3%,the base directed concentration causes largely increase of charged amino acid,and this phenomenon possibly has vital significance to the in vitro evolution of thermo-stability enzyme.4.In order to ensure the usability of low-fidelity enzyme Taq-T664R in the in vitro evolution,this research took endo-cellulase,which play an important role for lignocelluloses degeneration process, as the target,carried on in vitro mutagenesis by using Taq-T664R mutant,and obtained one high-activity mutant by selection,its relative activity was 1.8 times of wild type.