Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification
Research article published in Molecular medicine reports (2015)
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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Zusammenfassung
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.
Warum dies für die Hirudotherapie relevant ist
This study reports site-specific incorporation of boronophenylalanine into recombinant hirudin at position 63 using an engineered Escherichia coli expression system, with the goal of enhancing bioactivity. The modified hirudin showed greater antithrombin activity against human thrombin and stronger inhibition of fibroblast L929 proliferation compared with unmodified recombinant hirudin. For ASH's domain, this work is relevant as a protein-engineering effort to improve the potency of hirudin, a substance indexed under 'Hirudins' and central to ASH's scope of interest. The caveat is that these are preclinical in-vitro findings only; no in-vivo, pharmacokinetic, safety, or clinical data are presented, and the functional significance of the fibroblast-proliferation effect is not established.
Zitation
Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification
Xin X et al. · Molecular medicine reports, 2015
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