Recombination-engineered E. Coli Chromosomal Gene Knock-in And Expression

With the development of molecular biology,recombineering based homologous recombination engineering has become a common technology,especially used in Escherichia coli.The high efficiency,short homologous sequencerequired and convenient operation process make the homologous recombination engineering more and more popular.Combine with the homing endonuclease I-Scel,markerless gene deletion can be achived.It is the insoluble heterologous protein that brings many difficulties to research the function of the gene and the protein for us.To solve this problem we can increase the production of the fusion protein that includes fusion tag and target protein.It can increase expressed production and soluble part of the target protein by co-expressing the fusion tag.As we all know,the maltose binding protein(MBP)is the most common used tags.Derived from the Nla protease(Nuclear inclusion a protein)of tobacco etch virus,TEV protease have high specificity and shearing activity.It is the first choice to cut the affinity tag from fusion protein.Fusion tag and target protein are separated if enzyme digestion site of TEV in the middle of both is cut.The operation is very convenient.But the TEV protease is very expensive.Some methods were used to construct a strain LS2401 which expressed the TEV protease in the second chapter of this research.First,we constructed a clone which included the enzyme digestion site of TEV protease to link the gene of MBP with TEV protease.At the same time,the strong promoter T7 and chloramphenicol resistance genes were successfully introduced.Then the malE gene in the E.coli BL21(DE3)was replaced by this fragment of the clone by Red/ET homologous recombination.That the enzyme digestion site between the gene of TEV protease and the gene of maltose binding protein was cut by TEV protease liked a scissors released TEV which is subsequently isolated through Ni-NTA affinity purification.The shearing efficiency of this system can reach above 80%?Meanwhile the activity and yield of TEVprotease that this cystem produced is high.This cystem can bring a lot of convenience to the research of laboratory.TEV protease cut the protein which the chromosome expressed rather than the target protein if the system was applied to cut fusion protein in the cell.So this system should be modified due to its low efficiency.Next two strains that express TEVprotease were constructed.Homologous recombination was used to remove the enzyme digestion site of TEV protease in the middle of MBP and TEV.The homologous sequence we used is 50 bp and 500 bp.We also use the homing endonuclease I-SceI to help this process succeed.However,none worked.Then the same methodwas used to replace the malE gene in theE.coli BL21(DE3)as the second chapter.The recognition site of TEV protease was removed.In order to verify the feasibility of this method,two plasmids that contain fusion tags and target protein were transformed into this system by the electrotrans.Under induction,genome and plasmid expressed protein showed the same time profile.TEV protease that the genome expressed cut the fusion protein.The efficiency of enzyme digestion can reach above 70%.Finally the hitagged target protein can be isolated through Ni-NTA affinity purification.As atool,it is very convenience to use in the experiment.Traditionally,the purification of target protein needs twice.If we use this system in the research,the purification of target protein only needs once.Meanwhile it can be reduce the cost of scientific research for us.