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Gene Synthesis> | Custom oligonucleotides were purchased from Merck Sigma Aldrich. Buffers, nucleotide triphosphate, and PrimeSTAR® HS DNA polymerase were purchased from Takara. All restriction enzymes were purchased from ThermoFisher Scientific. NEBuilder® HiFi DNA assembly master mix was purchased from New England Biolabs. A gene with codons optimized for expression in E. coli, encoding a protein composed of a Nterminal hexa-His tag, the 76 amino-acid residues of human ubiquitin and residues 24 – 474 of OaAEP1 (C247A) was inserted into the coding region (NcoI-NdeI) of the pET-28b (+) vector (Genscript). | Get A Quote |
Asparaginyl endopeptides (AEP) are recognized for their catalytic efficiency, presenting as ideal tools for protein bioconjugation. However, the peptide ligation catalyzed by AEP is reversible. In an attempt to obtain high reaction yields, thiodepsipeptides have been used as substrates but found to be highly unstable, and labeling is only limited to the N-terminus. To maximize the potential use of AEP, here we developed a novel chemo-enzymatic sequence for protein bioconjugation at both the N- and C-termini. In this system, an alternative recognition sequence, Asn-Cys-Leu, was used. Upon ligation, the reaction yields Cys-Leu as leaving group, and its reactive 1,2-aminothiol functionality was quenched by an effe... More