Abstract: To optimize the orthogonal Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)/tRNA system for site-specific incorporation of p-azido-phenylalanine (pAzPhe) in E. coli.The human endostatin mutant (mhEn) with pAzPhe inserted at specific amino acid site was expressed using the optimized system.The promoter of the orthogonal MjTyrRS was mutant to TTAA from TATAA at the -15 position.The Asp at 286 amino acid site of the orthogonal MjTyrRS was mutated to Arg to increase the recognition of the anticodon (CUA) in the amber suppressor tRNA of MjTyrRS.The recombinant plasmids pST and pET32a-mhEn were co-transformed into DH10BDE3.The mhEn was expressed in the medium containing pAzPhe after IPTG induction and the mhEn was purified by Ni-affinity chromatography with a purity of 90%.The optimized orthogonal MjTyrRS/tRNA system could incorporate pAzPhe with higher efficiency and the yield of mhEn was improved significantly.