title: Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification.
abstract: To improve the bioactivity of recombinant (r)Hirudin, the orthogonal pair MjBTyrRS/tRNATyr cua (made up of the boronophenylalanine, tRNA and tRNA synthetase), was selected to incorporate boronophenylalanine site‑specifically into rHirudin at the 63 sites in an Escherichia coli system in response to the TAG codon. Following fusion with the gIII signal peptide and a hexahistidine tag, the modified protein was secreted into Luria‑Bertani culture medium and purified by nickel-nitrilotriacetic acid affinity chromatography following a gel filtration column. In a 200 ml flask, the yield of boronophenylalanine‑modified hirudin was 10 mg l‑1 and that of rHirudin was 19 mg l‑1. The authenticity of the purified proteins was verified using matrix-assisted laser desorption ionization time of flight mass spectroscopy and antithrombin activity assays. The results revealed that the antithrombin activity of the boronophenylalanine‑modified hirudin to human thrombin was more enhanced than that of rHirudin. The modified hirudin demonstrated stronger proliferation inhibiting ability on fibroblast L929 cells compared with that of rHirudin.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Xin
authors.affiliation: Department of Chemistry, Texas A&M University, College Station, TX 77840, USA.
authors.forename: Wenshe
authors.initials: W
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors.forename: Liyun
authors.initials: L
authors.lastname: Sun
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D006629:Hirudins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005343:Fibrinolytic Agents; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine