Systematic Optimization On Overexpression Of Butelase-1 In Escherichia Coli And It’s Application

Butelase-1 is an asparagine endopeptidase peptide ligase derived from Clitoria ternatea.It has been used in the cyclization or ligation of peptides or proteins and labeling of bacteria with the advantages of simple recognition sequence and high catalytic efficiency.In order to improve the activity and biomass of recombinant Butelase-1 expressed in Escherichia coli,a variety of biotechnology methods were used to transform the expression plasmid and screen the expression strain,which significantly improved the activity of the recombinant Butelase-1zymogen after activation.It was applied to the head-to-tail cyclization of the food-derived anticancer peptide Lunasin,and the changes in the anticancer activity and stability after the cyclization of Lunasin were discussed.The main research contents are as follows:（1）The yield of active Butelase-1 was improved by screening suitable host strains,co-expressing molecular chaperones and optimizing recombinant expression conditions.Based on the Butelase-1 recombinant expression vector constructed in the laboratory in the early stage,the host strain was optimized.When the host strain E.coli Rosetta gami B（DE3）was replaced with E.coli SHuffle T7,the activity of Butelase-1 would be significantly improved.The induction and expression conditions of recombinant protein were further optimized,the enzyme activity yield can reach 1796.45 U/L fermentation broth.At the same time,four molecular chaperone expression vectors and Butelase-1 recombinant plasmids were co-expressed in E.coli,and it was found that Dsb C protein could significantly increase the activity of Butelase-1.（2）Synonymous rare codon substitutions improved the yield of Butelase-1.In view of the possible reason that the insufficient folding of the recombinant protein of Butelase-1 zymogen restricts the enzyme activity of Butelase-1,the Valine of positions 85,158,187,237 and 385located on theα-helix andβ-sheet of the Butelae-1 three-dimensional structure were selected.One or more preferred codons（GUG）of the E.coli expression systems were synonymously mutated to rare codons（GUA）,which improved the correct folding ratio of the zymogen.After replacing Val85,Val158,Val187,and Val237,the enzyme activity yield of Butelase-1 was the highest at 5059.32 U/L fermentation broth.（3）Optimized the regulation of Butelase-1 zymogen expression by RBS and AS.Nine kinds of RBS with different strengths were designed and corresponding expression vectors were constructed.The results showed that the activity of Butelase-1 regulated by RBS7 was the highest.Five kinds of AS with different lengths were further designed and corresponding expression strains were constructed.The results showed that the newly designed five kinds of AS caused the decrease of the enzyme activity and yield of Butelase-1 to varying degrees.After serial optimization of strains and plasmids,the yield of recombinant zymogen and the yield of activated Butelase-1 were 195.20 mg/L fermentation broth and 19.25 mg/L fermentation broth,respectively,and the enzyme activity and the specific enzyme activity of Butelase-1 were 8183.54 U/L fermentation broth and 386.93 U/mg,respectively.（4）It was determined that the kinetic parameters of the recombinant Butelase-1 enzyme were basically the same as those of the natural Butelase-1 enzyme.The Michaelis constant,turnover number and catalytic efficiency index were K_m=229.89μmol/L,k_cat=2.53 s^-1,k_cat/K_m=11048 M^-1 s^-1.The recombinant Butelase-1 was used to successfully cyclize the food-derived anticancer peptide Lunasin head-to-tail with a yield of 91.7%.After isolating and purifying the cyclic peptide,its anticancer activity was determined,and it was found that the activity and the stability of Lunasin increased by about 40%and 60%after cyclization,respectively.