Preliminary Study And Establishment Of TEV Protease Activity Analysis System

Tobacco plaque virus protease(TEV)is a catalytic region by a molecular weight of 27 kDa in the protein,encoded by tobacco leaf spot virus.It is a Cysteine protease with high specificity,Because of the specificity of the sequence of action,It has been widely used to exscind fusion tag.The protease recognizes cleavage sites of glutamic acid-asparagine-leucine-tyrosine-phenylalanine-glutamine-glycine and cleaves bonds of peptide between glutamine and glycine.Because of its high specificity and activity rate,and can maintain high enzymatic efficiency in different reaction buffers,TEV protease is widely used in the expression and purification of recombinant protein.The purpose of the reserch design is to obtain a substrate which can be specifically cleaved by TEV protease digestion recognition substrate through basic construction substrate and blue white spot experiment.The analysis of tev protease activity is promoted from a semi-quantitative and imperfect level to an Quantitative analysis method which can be used from a quantitative angle and thinking through data.Not only lays the foundation for the detection of tev enzyme activity,but also lays the foundation for the establishment of quantitative analysis system of tev protease activity.At the same time,this indirect complementary experiment of a peptide lays the foundation for determining the activity of other proteases.In this study,through constructed a series of vectors pQE2-MBP-α 、pQE2-MBP-α-ACP、pQE2-DsbA-α-ACP、pBAD24M-DsbA-α-ACP、pBAD24M-DsbA-αand so on.Mading high expression of protein and analysis of tev restriction enzyme in vitro,carrieding a peptide complementation LacZ(a gene encoding β-galactosidase in the coding region of lactose operon)in strains DH5α and JM109.TEV peptide cleavage vector releases a small peptide,Peptide a is complementary to LacZ Δ M15,a large fragment LacZ(β-galactosidase)-deficient strains such as DH5α and JM109.Bacteria produceβ-galactosidase with catalytic activity and beta-galactosidase hydrolyzes substrate X-gal(5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside)to produce color.Finally,We can find a specific tev protease substrate.Finally,through a series of blue and white spot experiments,pBAD24M-DsbA-αvector is successfully screened as a substrate which can be specifically recognized by tev.Through a series of blue and white spot experiments,DsbA-α protein is successfully screened as a substrate which can be specifically recognized by tev.It can accumulate stably in Escherichia coli.when expressed alone,blue white spot screening is white,and when co-expressed with tev enzyme,blue white spot screening is blue.When co-expressed with different active TEV(MBP-TEV、MBP-Ybbr-TEV、MBP-ACP-TEV).The time when blue colonies appeared was different.The higher the activity,the shorter the occurrence time of blue spot.The results showed that this paper established a sensitive method for the analysis of tev protease activity.