Enhancing Soluble Expression,Activity And Stability Of The Tobacco Etch Virus Protease And Its Immobilization On Cellulose

In the process of protein biological expression,peptides of different tags?such as his-tag,flag-tag?and proteins?such as GST and MBP?often need to be fused to the C-terminal or N-terminal of the target protein to improve the expression yield,solubility of the target protein and achieve protein purification.However,fused peptides and proteins often need to be cut off from the target protein.Currently,the only biocompatible protein splicing method is enzymatic digestion,although a variety of mammalian enzymes have been used for digestion,such as factor Xa,enterokinase and?-thrombase.However,it has been reported that in some specific cases these enzymes are not highly specific and have off-target effects.Tobacco Etch Virus protease?TEVp?,with its good activity and sequence specificity,has been highly valued in application.In addition,TEVp is the only protease that combines multiple useful characteristics of biotechnology applications,from simple and economical production to availability of open source vectors and mutants for a variety of in vitro and in vivo applications.The expression of TEV protease in escherichia coli is mainly in the form of inclusion body and is self-digest,which limits its application range.At present,in order to improve the solubility and stability of TEVp,many methods are used to improve the expression of TEVp in escherichia coli,such as codon optimization,solubilization labeling and site-directed mutation.However,the effects of several common soluble tags are not perfect.For example,although MBP could improve the solubility of TEVp,it could not improve its expression level.Trx tag did not improve the expression level and solubility of TEVp significantly.Although the expression level of GST fusion did improve,it could not promote the solubility of TEVp.Therefore,in this study,we first expected to find a more appropriate tag,which not only improve the expression of TEVp in escherichia coli,but also improve its solubility.In order to improve the activity of TEVp,many mutants of TEVp have been reported.In vitro application,one of the reported effective mutants is TEVp^6M?T17S/L56V/N68D/I77V/S135G/S219V/?238?.In the application of eukaryotic cells,two TEVp with better activity have been reported recently.One of them was a TEVp mutant?secTEVp-QSG?that introduced mutations at two N-glycosylation sites of TEVp and an exposed cysteine:Asn23Gln,Thr173Gly and Cys130Ser.In vivo experiments showed that the mutant had high activity in endoplasmic reticulum.The other is a TEVp mutant with higher enzyme digestion activity witch was found through multiple mutation screening in yeast cells,and its enzyme digestion efficiency is four times higher than that of ordinary TEVp,which is named as TEVp-Fast.In TEVp-Fast,in addition to the Ser219Val mutation site that avoids self-digestion,there are two other point mutations:Gly79Glu and Thr173Ala.In order to obtain mutants with higher enzyme cleavage activity,this study hopes to determine whether these sites can also promote the activity of TEVp in vitro.In addition,in the process of use,we found that TEVp easily deactivation,Long time storing at-80?will also be disabled.In order to improve the stability and utilization rate,this study hopes to improve the stability and expand the application range of TEVp by immobilizing TEVp.This experiment focused on the above three aspects and obtained the following results:?1?We used CBD as the soluble tag of TEVp.It was expressed in large quantities in escherichia coli compared with the MBP tag,It not only increased the expression amount,but also increased the solubility of TEVp,and finally increased the yield of TEVp.?2?OnthebasisoftheexistingTEVp^6M?T17S/L56V/N68D/I77V/S135G/S219V/?238?in the laboratory,we first introduced Thr173Ala through point mutation,and then introduced Gly79Glu and Cys130Ser separately or simultaneously to obtain three new TEVp mutants:TEVp^7ME,TEVp⁷MSMS and TEVp^9M.The activity and stability of the mutant and TEVp^6M were compared.We found TEVp^9M at 4?and 25?has better activity;at 25?and 37?has better stability.?3?As an affinity tag,CBD can be combined with cellulose,so TEVp can be immobilized through CBD.The results showed that the activity of immobilized CBD-TEVp was slightly lower than that of free-state TEVp,but the efficiency of TEVp could be improved by increasing the number of using times.?4?In general,the addition of biological enzymes will increase the purification steps of subsequent removal.The immobilized TEVp can achieve one-step removal of affinity tags and the acquisition of target proteins,which is both efficient and convenient.