Enhancement Of Polymerase Activity Of Thelarge Fragment Indna Polymerase I From Geobacillusstearothermophilus By Site-directed Mutagenesis At The Active Site

According to the amino acid sequence and the three-dimensional structure,DNA polymerase can be divided into 6 different families,namely,A,B,C,D,X and Y family.Bst DNA polymerase derived from Bacillus stearothermophilusbelongs to the family A which consists of three independent domains consisting of(I)5'-3'exonuclease domain(II)5'-3'polymerase domain(III)3'-5'exonuclease domain.The large fragment of DNA polymerase I with 5'-3'DNApolymerase activity while in absence of 5'-3'exonuclease activity possesses high thermal stability,chain displacement activity and polymerase activity.Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification(IMSA)which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases.However,the current market Bst DNA polymerase is limited by international patents such as NEB patent restrictions,the price is more expensive than the general nucleic acid amplification enzyme,so there is an urgent need to develop a new and more effective Bst DNA polymerase to meet the growing increased market demand.In this study,the Bst DNA polymerase gene was extracted from a new strain Bacillus stearothermophilus GIM1.543,and the recombinant plasmid pET21a-Bst was constructed and transformed into the host.The expression of Bst DNA polymerase was successf?Lly achieved.The mutant was constructed by RF cloning method,and the mutant was constructed by using the Bst DNA polymerase structure and the active site predicted by homology modeling.Four mutations were selected from the four active sites(Gly310,Arg412,Lys416,Asp540)Its expression was purified with Ni column affinity chromatography to obtain high purity protein.At the end of this study,the VP1 gene of EV71 was used as template,and the expression of G310 L,G310A and D540 E were detected by IMSA color determination method,thermostable fluorescence method and ion pair anti-high performance liquid phase method.The mutant protein exhibits superior to commercial Bst DNA polymerase in isothermal amplification.This study significantly improvedthepolymerase activity of the large fragment in DNA polymerase I from Geobacillus stearothermophilus by site-directed mutagenesis at the active site and break the limits of foreign patents.It provides a theoretical basis and idea for the optimization of Bst DNA polymerase,which has certain scientific significance and social benefit.