Selection conditions of the recombinant bovine α-interferon from E. coli inclusion bodies

Alexander Zhydzetski ,M I Patapovich,U A Prakulevich,I V Kudina ,М В Шолух

doi:10.29235/1029-8940-2019-64-1-7-17

Alexander Zhydzetski , M I Patapovich + Show 3 more

Open Access

https://doi.org/10.29235/1029-8940-2019-64-1-7-17

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Abstract

Like other proteins of the cytokine family, bovine α-interferon activates and modulates antiviral state of the target cells and inhibits division and growth of the infected cells which makes it an excellent candidate as a new antiviral therapeutic agent.This study is concerned with the determination of the optimal isolation, purification and refolding conditions of the recombinant bovine interferon-α (rbIFN-α) from inclusion bodies (IBs). Main methods used were UV/Visible spectroscopy, electrophoresis, liquid chromatography and refolding by dilution.It was found that two step IBs washing with solutions containing 50 mmol/l Tris, 50 mmol/l NaCl and 3.5 mol/l urea and their subsequent solubilization in 50 mmol/l Tris-HCl, pH 9.8 mol/l Urea and 20 mmol/l β-mercaptoethanol allow us to receive the target protein in monomeric form and 53.18 ± 9.3 % purity. Further application of the anion-exchange tandem chromatography on DE 52 cellulose and toyopearl DEAE-650 M gives a possibility to remove the major impurities and obtain rbIFN-α with 80.7 ± 8.6 % purity. Refolding by dilution in the buffer containing 20 mmol/l NaPB, рН 7.4, 0.4 mol/l sucrose, 1 mmol/l L-Cys, 0.1 mmol/l L-Cystine, 1 mmol/l EDTA, 0.05 % Kolliphor EL at 10 °C followed by the protein collection allows to get the recombinant rbIFN-α in homogeneous state, with 98.43 % purity and antiviral activity about (5 ± 3.6)•106 U/mg.

Highlights

It was found that two step inclusion bodies (IBs) washing with solutions containing 50 mmol/l Tris, 50 mmol/l NaCl and 3.5 mol/l urea and their subsequent solubilization in 50 mmol/l Tris-HCl, pH 9.8 mol/l Urea and 20 mmol/l β-mercaptoethanol allow us to receive the target protein in monomeric form and 53.18 ± 9.3 % purity
Несмотря на то что в настоящее время существует целый ряд методических подходов к рефолдингу рекомбинантных белков, до сих пор нет универсального, позволяющего получить любой белок в активной форме
Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies / S

Summary

Introduction

It was found that two step IBs washing with solutions containing 50 mmol/l Tris, 50 mmol/l NaCl and 3.5 mol/l urea and their subsequent solubilization in 50 mmol/l Tris-HCl, pH 9.8 mol/l Urea and 20 mmol/l β-mercaptoethanol allow us to receive the target protein in monomeric form and 53.18 ± 9.3 % purity. Эффективность экстракции rbIFN-α из ТВ, чистоту белковых фракций и содержание мономерной формы целевого белка оценивали методом SDS-электрофореза в 15 %-ном полиакриламидном геле [14]. Такой этап необходим для удаления небелковых примесных соединений, в основном нуклеиновых кислот (НК) и липидных компонентов мембраны, а также клеточных белков, попадающих в ТВ, так как показано, что они снижают выход рефолдинга целевого продукта [18, 21].

Results

Conclusion