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Evaluation and comparison of affinity chromatography and precipitation– based methods on purification of recombinant streptokinase
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Masoud Seyedinkhorasani1    , Malihe Keramati2 , Reza Ahangari Cohan3 , Farzin Roohvand4 , Dariush Norouzian 5 |
1- PhD Student of Pharmaceutical Biotecnology, Nano-biotechnology Department, Pasteur Institute of Iran (PII), Tehran, Iran
2- Assistant Professor, PhD of Pharmaceutical Biotechnology, Nano-biotechnology Department, Pasteur Institute of Iran (PII), Tehran, Iran
3- Associate Professor, PhD of Pharmaceutical Biotechnology, Nano-biotechnology Department, Pasteur Institute of Iran (PII), Tehran, Iran
4- Associate Professor, PhD of Medical Biotechnology, Virology Department, Pasteur Institute of Iran (PII), Tehran, Iran
5- Professor, PhD of Biochemistry, Nano-biotechnology Department, Pasteur Institute of Iran (PII), Tehran, Iran , dnsa@pasteur.ac.ir |
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Abstract: (2556 Views) |
Background: Increase of protein purity is a serious challenge in the production of recombinant therapeutic proteins. For this purpose, several strategies have been employed to purify the target protein, among which the affinity chromatography-based purification methods and tagged proteins such as Ni-NTA are common and but costly. Therefore column-free purification techniques, such as using elastin-like proteins (ELPs), are being developed as alternative method. In present study, the efficacy of Ni-NTA, ELP, and the combined Ni-NTA/ELP was evaluated for purification of streptokinase as a model protein at lab scale.
Materials and methods: Streptokinase gene was amplified by PCR, cloned into pET21-ELP, and transformed into E.coli BL21 Rosetta cell. The expressed protein was then purified using the methods described above. The identity and purity of the protein were evaluated by western blot using Anti-His antibody and SDS-PAGE, respectively. The purification yield was also calculated for each method by micro-Bradford assay.
Results: The purification yield was calculated as 76 ± 16 μg/ml in ELP precipitation, 108 ± 14 μg/ml in Ni-NTA purification and 88 ± 11 μg/ml in Ni-NTA/ELP combined method, respectively. The SDS-PAGE results showed that the purity of streptokinase purified by the combined method was higher than that of each method separately.
Conclusion: The present study showed that the integration of purification methods in one step can increase the purity and yield in purification process of recombinant therapeutic proteins. |
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| Keywords: ELP, Elastin like polypeptide, Protein purification, Column-free purification, Streptokinase |
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Full-Text [PDF 462 kb]
(3582 Downloads)
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Semi-pilot: Experimental |
Subject:
biotechnology
Received: 2019/09/1 | Accepted: 2020/01/27 | Published: 2020/12/20
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