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Soluble Expression of Recombinant Human Cystatin C and Comparison of the Ni Column and Magnetic Bead Purification

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Abstract

Cystatin C, also known as γ-trace or post-γ-globulin, is a cysteine protease inhibitor from the cystatin superfamily. It is usually used as a marker of the glomerular filtration rate owing to its low molecular weight and constant secretion. The recently available methods for cystatin C preparation have low outputs. Hence, a productive preparation system is urgently required. In this study, a 6 × His-tag coupled with a thrombin cleavage site was fused to the C-terminus of cystatin C, and the protein was well expressed in Escherichia coli after optimization. Then, two different systems were used to obtain no-tag cystatin C: a traditional nickel (Ni)-column system and a subtly Ni magnetic bead system. The column system was more commonly used, and the magnetic bead system was more convenient. Cystatin C (purity > 97%) was successfully obtained, and the yields in both the systems were higher than those in previous studies. Further, the proper folding status and bioactivity of recombinant cystatin C were confirmed using the papain inhibition assay, dynamic light scattering, and circular dichroism spectroscopy.

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Abbreviations

CD:

Circular dichroism

DLS:

Dynamic light scattering

GFR:

Glomerular filtration rate

LB:

Lysogeny broth

iPCR:

Inverse polymerase chain reaction

IPTG:

Isopropyl-β-d-thiogalactoside

Magbeads:

Magnetic beads

MBP:

Maltose-binding protein

PBS:

Phosphate-buffered saline

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No. 21375039), the Shanghai Committee of Science and Technology (Grant Nos. 16142202000 and 18142202600), the Research Program of Shanghai Chemical Reagent Research Institute Co., Ltd. (Grant F100-81707), and the National Special Fund for State Key Laboratory of Bioreactor Engineering (Grant No. 2060204).

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  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China

    Yibin Zhang, Jian Zhao, Shiyu He & Xuni Cao

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  1. Yibin Zhang
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Zhang, Y., Zhao, J., He, S. et al. Soluble Expression of Recombinant Human Cystatin C and Comparison of the Ni Column and Magnetic Bead Purification. Protein J 39, 85–95 (2020). https://doi.org/10.1007/s10930-019-09873-0

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