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.

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

“…Xin et al reported that the antithrombin activity of rH was significantly increased after modified with boronophenylalanine. In addition, the proliferation inhibiting ability on fibroblast L929 cells was also obviously increased, which indicated that boronophenylalanine-modified hirudin might be used as a promising agent to treat fibrosis (Xin et al, 2015). Furthermore, rhSOD2-hirudin, a dual-feature fusion protein, fused by Hirudin and human manganese superoxide dismutase (hSOD2) could inhibit the proliferation of fibroblasts and reduce the Hydroxyproline (HYP) production in vitro by inhibiting the activity of thrombin.…”

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis

“…Even more strikingly, TH lacks the elongated C-terminal tail that is characteristic for all hirudins (Salzet 2000 ). The C-terminal tail of hirudin blocks the fibrinogen-binding site of thrombin, the so-called exosite 1 (Maraganore and Fenton 1990 ; Xin et al 2015 ; Priestle et al 1993 ). In boophilin, ornithodorin and rhodniin, the C-terminal second globular domains adopt these functions (Macedo-Ribeiro et al 2008 ; Mans et al 2002 ; Friedrich et al 1993 ).…”

“…Even more strikingly, TH lacks the elongated C-terminal tail that is characteristic for all hirudins (Salzet 2000). The C-terminal tail of hirudin blocks the fibrinogen-binding site of thrombin, the so-called exosite 1 (Maraganore and Fenton 1990;Xin et al 2015;Priestle et al 1993). In boophilin, Fig.…”

Make it double: Identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis

“…Even more strikingly, TH lacks the elongated C-terminal tail that is characteristic for all hirudins (Salzet 2000). The C-terminal tail of hirudin blocks the brinogen-binding site of thrombin, the so-called exosite 1 (Maraganore and Fenton 1990;Xin et al 2015;Priestle et al 1993). In boophilin, ornithodorin and rhodniin the C-terminal second globular domains adopt these functions (Macedo-Ribeiro et al 2008; Mans et al 2002;Friedrich et al 1993).…”