Expression,purification And Functional Analysis Of Asparaginyl Endopeptidase Cyclase In Different Hosts

Asparaginyl endopeptidases(AEPs)is a key enzyme that catalyzes the formation of cyclotides,which is widely distributed in plants.Cyclotides have a cyclic backbone and a typical cysteine knot consisting of six conserved cysteine residues that form three disulfide bonds.Due to such an extremely stable structure,the cyclotides show great potentials to be applied as backbones for drug design and thus have attracted tremendous attentions.Furermore,many engineered active peptides would be stabilized for longer half-life via cyclization by AEPs.However,most of the AEPs are currently extracted from plant tissues,being a tedious process but giving low enzyme yields.The most straightforward and efficient way to overcome these restrictions would be producing the enzymes recombinantly through the highly efficient microbial expression systems.To this end,in this study,an AEP(Oa AEP1_b)cyclase encoding gene was cloned from Oldenlandia affinis and expressed in Escherichia coli and Pichia pastoris,respectively.For the expression in E.coli,a series of tags,including maltose binding protein(MBP),N-utilization substance A(Nus A),small ubiquitin modified protein(SUMO),green fluorescent protein(GFP),and ubiquitin(Ub),were individually fused with the AEP cyclase,and the effect of each tag on increasing the expression of AEP was evaluated.Among them,MBP significantly increased the yield of AEP cyclase,reaching 0.36±0.05 mg/m L.Meanwhile,based upon codon optimization,the AEP-encoding gene was integrated into the genome of P.pastoris for secretory expression.We attempted integration of various copies of the AEP-expressing cassette and found that the expression level of the target protein was positively linked with the copy number until it reached 3.The highest titer was ca 0.89±0.03 mg/m L,which were much higher than that in E.coli.Continued addition of gene copy did not significantly increase the expression of the AEP cyclase.In vitro biochemical analysis confirmed that the exogenously expressed AEP cyclase could efficiently cleave and cyclize different engineered substrates that contain target sequences.Thus,this study has laid a solid experimental foundation for using AEP cyclase as an efficient toolkit for both research and practical applications in peptide engineering.