Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification. Academic Article uri icon

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 sitespecifically 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 LuriaBertani culture medium and purified by nickel-nitrilotriacetic acid affinity chromatography following a gel filtration column. In a 200 ml flask, the yield of boronophenylalaninemodified hirudin was 10 mg l1 and that of rHirudin was 19 mg l1. 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 boronophenylalaninemodified 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.

published proceedings

  • Mol Med Rep

author list (cited authors)

  • Xin, X., Liu, W., & Sun, L.

citation count

  • 2

complete list of authors

  • Xin, Xiujuan||Liu, Wenshe||Sun, Liyun

publication date

  • May 2015