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Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein

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Abstract

A purification system was constructed with the N-segment of the Npu DnaE split intein as an affinity ligand immobilized onto an epoxy-activated medium and the C-segment used as the cleavable tag fusing target protein. The affinity properties of C-tagged proteins adsorbed on IN affinity chromatography medium were studied with GFP as a model target protein. The saturated adsorption capacity and dynamic adsorption capacity reached 51.9–21.0 mg mL−1, respectively. With this system, two model proteins, GFP and alcohol dehydrogenase (ADH), has been successfully taglessly purified with regulation of Zn2+ and DTT. The yield, purification factor and purity of purified tagless GFP reached 39, 11.7 and 97%, respectively; while these values for purified tagless ADH were 38.2, 6.8 and 91%, respectively. These results showed that the system for Npu DnaE split intein-mediated affinity adsorption and in situ cleavage is a potential platform for recombinant protein production.

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Acknowledgements

We thank Prof. Zhilei Chen (Texas A&M University) for help in troubleshooting advice. This work was supported by the National Key R&D Program of China (Grant No. 2018YFC1603202/2018YFC1603200), the 111 Project (No. 111-2-06); and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Hai-Feng Xia, Ting-Jun Zhou, Ye-Xing Du & Yu-Jun Wang

  2. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Hai-Feng Xia

  3. Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, 43210, USA

    Chang-Hua Shi & David W. Wood

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  1. Hai-Feng Xia
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  2. Ting-Jun Zhou
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  5. Chang-Hua Shi
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Correspondence to Hai-Feng Xia.

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Xia, HF., Zhou, TJ., Du, YX. et al. Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein. Bioprocess Biosyst Eng 43, 1931–1941 (2020). https://doi.org/10.1007/s00449-020-02382-w

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  • DOI: https://doi.org/10.1007/s00449-020-02382-w

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