Recombinant Expression And Activity Study Of Several Prokaryotic DNA Helicases

Helicase is an enzyme that hydrolyzes ATP to supply energy to untie the hydrogen bond of DNA or RNA.It plays a role in almost all processes involving nucleic acid in cells.Helicase can be used in polymerase chain reaction(PCR)to construct multi enzyme reaction system,DNA sequencing technology and other biological research.The helicase genes tk0178,ma1411 and tete0604 were isolated from Thermococcus kodakarensis,Methanosarcina acetivorans and Thermoanaerobacter tengcongensis,respectively.At present,the commercial helicase still has some shortcomings in the aspects of helicase activity and reaction temperature.Therefore,it is very important to find new DNA helicase to enhance the application of helicase.In this paper,the helicase genes tk0178,ma1411 and tete0604 were cultured,cloned and expressed in archaea;the proteins encoded by the helicase genes tk0178 and tete0604 were purified by heat treatment and anion exchange column combined purification strategy,and the ATPase activity was determined;finally,the purified helicase was applied in PCR reaction to reduce the non specificity of thermal cycle PCR Amplification and construction of helicase dependent isothermal PCR system.In this paper,the proteins encoded by helicase TK0178,MA1411 and TTE0604 genes were successfully expressed.Helicases TK0178 and TTE0604 were expressed in the supernatant with the size of 76 k Da and 82 k Da,respectively,and the expression of the target protein in the supernatant was similar to that of the total protein,while the helicase MA1411 was mainly expressed in precipitation.After purification,high purity helicase TK0178 and TTE0604,protein electrophoresis showed almost only one band.Both purified TK0178 and TTE0604 had ATP enzyme activity.In the presence of single-stranded DNA,the TK0178 helicase ATP enzyme activity increased with the increase of temperature,and the highest activity was 0.387 U/mg at 100 ℃.When the temperature was lower than 60 ℃,there was almost no activity.The activity range of TTE0604 helicase ATP is narrow,and the specific activity can only be measured at60 ℃ ～ 65 ℃.At 60 ℃,the specific activity is 0.134 U/mg at 65 ℃,and the specific activity is 0.136 U/mg at 65 ℃.DNA helicase can improve the performance of thermal cycling PCR and isothermal PCR: helicase TK0178 can reduce the non-specific amplification of thermal cycling PCR,and nucleic acid electrophoresis shows that the non-specific amplification band almost disappears;helicase TK0178 can also cooperate with polymerase KOD pol in isothermal amplification to amplify specific DNA fragments in a short time(～ 1.5h).Compared with the commercial isothermal amplification kit,the medium and low GC content DNA template can only be amplified,and the isothermal amplification reaction system constructed with helicase TK0178 can specifically amplify the DNA template with high GC content.This study provides an experimental basis for the construction of PCR multi-enzyme reaction system with high activity,high specificity and high stability.