Oriented Immobilization Of TEV Protease For Visually Detecting On-resin Cleavage

Tobacco etch virus protease(TEVp)is widely applied for removal of the fusion tags due to its stringent sequence specificity and high catalytic efficiency.To further improve TEVp usuage efficiency,in this work,we applied the oriented immobilization of the TEVp variant with the N-terminal cellulose-binding module(CBM)on the regenerated amorphous cellulose(RAC).The constructed TEVp in the free and immobilized forms for cleaving the fusion protein were assayed.The four colored proteins as the visual tags for naked-eye detection of the on-resin cleavage mediated by the cognate TEVp were developed.The results are as follows:1.Construction of the fusion proteins.The active TEVp variant or the inactive TEVp variant with the N-terminal CBM tag or the C-terminal His6-tag were designed.The four colored proteins,including emerald green fluorescent protein EmGFP,red fluorescent protein mCherry,the brown protein maize sirohydrochlorin ferrochelatase(ZmSF)containing Fe-S cluster,and Vitreoscilla hemoglobin(VHb)containing the heme as the co-factor were attached with the CBM tag.A TEVp cut site was incoporated between the CBM and color proteins,except for two cut sites introduced between CBM and VHb.In addition,GST tagged Escherichia coli(E.coli)diaminopropionate ammonia-lyase(eDAL)or maize serine racemase(ZmSR)were used as TEVp substrate.2.Overexpression and purification of the fusion proteins.The expression plamids were transformed into E.coli BL21(DE3).After induction at 28 °C for 12 h,the cells were disrupted,and the supernatant containing the fusion proteins.TEVp was either purified by Ni-NTA agarose,or immobilized on the RAC.Other expressed CBM tagged proteins were also immobilized on the RAC.3.Analysis of the characters of the TEVp in the free or immobilized forms.After purification,the minor impurities were detected in the purified TEVp construct.The binding capacity was up to 220 mg TEVp construct per gram RAC.Kinetic analysis showed that the immobilized TEVp had less catalytic activity than the free one.After the protease was stored at 4 °C for 10 days,the activity was decreased 22 % for the immobilized protease and 60 % for the free protease.Approximately 2.1 % of the proteins were detached from RAC.After repetitive use for five times,the activity of the immobilized TEVp decreased to 40 %.4.On-resin cleavage of the fusion protein.Using the CBM tagged EmGFP assubstrate,the mass ratios of the immobilized protease to the fusion protein,and the reaction temperatures were optimized.With the ratio of the substrate to the protease was 30:1,and reacted at 35 °C for more than 3 h,the proteolysis process reached a steady state.5.Visual detection of the on-resin cleavage of the fusion proteins.After cleavage reaction,the different amounts of the colored proteins were detached from RAC,which were further confirmed by SDS-PAGE.The results also identified that on-resin cleavage efficiency and purity of the released target proteins are related with the context of the fusion tag,the incorporated linker composition,and the target protein.In conclusion,the fused TEVp was efficiently immobilized on RAC.The enhanced stability and repetitive use of the immobilized TEVp compensated slight loss of the catalytic efficiency toward the soluble protein substrate.On-resin cleavage of the fused colored protein is detected easily,simply and rapidly.Owing to low cost and high binding capacity of RAC,the TEVp immobilized on the resin is an ideal alternative for removing fusion tag.The developed naked-eye visible system is potentially used for tracking cleavage of fusion proteins on the other affinity resin mediated by the other sequence-specific protease.