The aim of the investigation was to develop the technique of recombinant protein biological activity recovery on affinity column during protein purification, the case of therapeutic cytokines: human interferon-alpha-2b (hIFN-α-2b) and human granulocyte-macrophage colony-stimulating factor (hGM-CSF). Recombinant cytokines was produced by bacterial producer (Escherichia coli). Proteins expressed were identified using ELISA and Western blotting methods. Proteins were present in the form of inactive inclusion bodies. To resolve this structure, we used denaturant conditions (6 M guanidinium chloride for hIFN-α-2b and 8 M urea for hGM-CSF ). A pre-washing of inclusion bodies fraction which allows to remove numerous ballast proteins. After a capture step with immobilized metal affinity chromatography (IMAC) a gradual refolding of proteins was performed on-column with a linear denaturant agents gradient from 6 M / 8 M to 0.5 M in the presence of reduced glutathione and oxidized glutathione. On-column refolding of hIFN-α-2b and hGM-CSF was developed for time and material costs reduction. The refolding efficiency reached 70%, whereas the target preparation purity was 95%. The biological activity of purified and refolded proteins was similar to those of other recombinant analogues. Obtained results suggest that there are some advantages of this technology in comparison with refolding by dialysis or dilution.

Цель исследования – разработка метода восстановления биологической активности рекомбинантного белка непосредственно на аффинной колонке в процессе очищения продукта на примере двух терапевтически значимых цитокинов: инрерферона-альфа-2b человека (чИФН-α-2b) и гранулоцитарно-макрофагального колониестимулирующего фактора человека (чГМ-КСФ). Рекомбинантные цитокины были получены из бактериального продуцента (Escherichia coli) в виде телец включения. Очистка белков осуществлялось посредством аффинной хроматографии. Для сокращения времени и материальных затрат был разработан метод проведения рефолдинга чИФН-α-2b и чГМ-КСФ, иммобилизованных на металл-хелатном сорбенте. Полученные результаты свидетельствуют о ряде преимуществ данной технологии в сравнении с рефолдингом посредством диализа или разведения.