Construction Of N-cysteine-free Npu DnaE Used In C-terminal Cleavage And Improving Proteolytic Stability Of Npu DnaE C-fragment

Background and aim:Intein is a part of polypeptide in the premature protein with the capability of self-splicing.Compared with continuous intein,split intein has the natural advantage of inhibiting uncontrollable early splicing and cleavage,and is often used in protein purification,protein connection,and toxin production.Npu DnaE,a kind of split intein from Nostoc punctiforme PCC73102,can meditate rapid and efficient protein trans-splicing.Some research introduced the D118G mutation in Npu DnaE and obtained a thiol compound-dependent C-terminal cleavage mutant.And they developed‘Split intein Mediated Ultra-Rapid Purification of Tagless Protein'(SIRP)with this Npu DnaE mutant,which made it of great potentiality in protein purification.The trans-splicing and C-terminal cleavage meditated by Npu DnaE intein are highly dependent on the catalysis of reducing agent.This may be related to the disulfide bonds in and between the Npu molecules formed by cysteine.In this study,the N-terminal cysteine-free mutants Npu N_C28SC59S-H and Npu N_C28IC59R-H were constructed,and the C-terminal cleavage reaction they meditated was studied.It's reported that Npu intein's C-fragment would be degraded during the expression and purification process,which reduced the yield and purity of recombinant protein and increased the cost of production.In this study,an N-terminally extended Npu C fragment,N2C,was constructed following the‘Capture and Collaspe'mechanism.Then the stability of N2C in prokaryotic expression system was studied,and the factors affecting N2C C-terminal cleavage reaction were also investigated.Method:In this study,we modified Npu N-fragment and Npu C-fragment and constructed recombinant expression plasmids with p ET28a and p ET30a vectors.We expressed the recombinant proteins in BL21(DE3),purified the proteins with affinity chromatography,concentrated proteins and replaced reaction buffer with Ion Exchange Chromatography(IEX)or ultrafiltration method.We conducted the C-terminal cleavage reaction after measuring the concentration of the proteins by Bradford method,and used SDS-PAGE and Western blot to investigate the stability of the Npu C fusion and the results of C-terminal cleavage reaction.Results:N-fragment cysteine-free Npu DnaE intein with purification tag on C-terminal,Npu N_C28SC59S-H and Npu N_C28IC59R-H,could mediate rapid C-terminal cleavage without reducing agent.And yield of C-terminal cleavage was 50%in 5 minutes,and 90%after 12 hours at 25 degrees Celsius without DTT.N2C significantly improved the stability of Npu C-fusion in the prokaryotic expression system and retained C-terminal cleavage activity and yield of C-terminal cleavage was 70%in 10 minutes and 90%in 30 minutes at 37 degrees Celsius with 1mmol/L DTT.The factors affecting the intein C-terminal cleavage reaction,such as temperature,the concentration of DTT and the ratio of N fragment and C fragment were investigated and C-terminal cleavage activity was less affected.