Enhanced And Evaluated The Thermal Stability Of RtULP1 ViaSpyTag/SpyCatcher Mediated Spontaneous Cyclization

The SpyTag/SpyCatcher cyclization technology cyclizes the target protein by cyclizing the protein backbone to increase the stability of the target protein,it based on a spontaneous and rapid ligation reaction.Studies identified that the cyclization was efficiently and spontaneously completed under a variety of environmental conditions,resulting in strong stability and maintaining enzyme catalytic activity and function.Small ubiquitin-like modifier(SUMO)is widely used as a label for soluble expression of recombinant protein.Ubiquitin-like-specific protease 1(Ulp1)is a major member of SUMO proteases,Which is responsible for both processing SUMO into its active form and deconjugating SUMO from its target proteins,however,its poor stability hampers its use in a wide range of applications.This study constructed a cyclized Ulp1 using SpyTag/SpyCatcher technology to obtain a ULP1 with better thermal stability,which provides the support for the widespread use of the SUMO tag.Here,recombinant truncated(rt)Ulp1(Gly304-Lys621)was used instead of Ulp1,which displays full proteolytic activity in cleavage reactions.Then we did optimization designs for escherichia coli codon bias and linear epitope,which dead against rtULP1 encoding nucleic acid sequence.Rt ULP1,Spy Tag-rtULP1-Spy Catcher(crtULP1),and Spy Tag(DA)-rtULP1-Spy Catcher(EQ)(lrtULP1)was designed and constructed.We compared the optimal reaction conditions and thermal stability of three proteases.Then we evaluated objective the influence of cyclic structure on the thermal stability of rtULP1 and the effect of SpyTag/SpyCatcher technology on the thermal stability of rtULP1.The results showed the optimal temperature of the crtUlp1 was 5°C higher than rtUlp1 and 10°C higher than lrtULP1.Furthermore,87.3% of crtUlp1 activity was retained upon incubation at 40°C for 2 h and 43.69% of lrtUlp1 activity was retained upon incubation at 40°C for 2 h,whereas rtUlp1 losted almost all activity.Moreover,the crtUlp1 also exhibiting high levels of activity at a wide range of p H values,whereas other two proteases only showed high levels of activity at their optimal p H and so on.These results indicated that SpyTag/SpyCatcher-mediated cyclization increased the stability of rtUlp1 and suggested that the technology represents a promising and effective strategy for enhancing rtUlp1 thermal stability.