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.
Resumen
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.
Por qué esto importa para la hirudoterapia
Este estudio informa sobre la incorporación sitio-específica de boronofenilalanina en hirudina recombinante en la posición 63 mediante un sistema de expresión en Escherichia coli modificado, con el objetivo de mejorar la bioactividad. La hirudina modificada mostró mayor actividad antitrombina frente a la trombina humana y una inhibición más potente de la proliferación de fibroblastos L929 en comparación con la hirudina recombinante no modificada. Para el dominio de la ASH, este trabajo es relevante como un esfuerzo de ingeniería de proteínas para mejorar la potencia de la hirudina, una sustancia indexada bajo 'Hirudinas' y central en el ámbito de interés de la ASH. La advertencia es que se trata únicamente de hallazgos preclínicos in vitro; no se presentan datos in vivo, farmacocinéticos, de seguridad ni clínicos, y no se establece la significación funcional del efecto sobre la proliferación de fibroblastos.
Citación
Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification
Xin X et al. · Molecular medicine reports, 2015
Contexto clínico relacionado
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026