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Cloning, expression in Escherichia coli, and purification of soluble recombinant duck interleukin-2

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Abstract

Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular and immunity of animals. In this study, a gene encoding duck IL-2 was cloned and the soluble recombinant duck IL-2 (rDuIL-2) was expressed in Escherichia coli via fusion with glutathione S-transferase (GST). The results indicated that the GST-rDuIL-2 fusion protein expressed in E. coli Origami (DE3) was confirmed to be of about 40 kDa molecular mass by SDS-PAGE and western blotting. In order to produce soluble rDuIL-2 in a low-cost, nontoxic and high-level expression process, lactose was used as a substitute for Isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the above recombinant strain Origami (pGEX-DuIL-2). By optimizing the expression conditions, the production of soluble GST-rDuIL-2 fusion protein was about 29% of total cellular soluble proteins, which was similar with IPTG used as inducer. The soluble GST-rDuIL-2 fusion protein was purified by one-step affinity chromatography, and GST was removed by thrombin. Then rDuIL-2 was purified by a second affinity chromatography. As a result, the 95% pure rduIL-2 was obtained, and the yield of rDuIL-2 was about 10.6 mg/l bacterial culture. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay in vitro. Our study provided a feasible and convenient approach to produce soluble and biologically active rDuIL-2, which would be used as an immunoadjuvants for enhancing vaccine efficacy.

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Acknowledgments

This study was supported by the Key Research Foundation of Fujian provincial department of Science and Technology, China (2009N0042) and the Fujian Provincial Nature Science Foundation (2010J01212).

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Authors and Affiliations

  1. School of Biotechnology Engineering, Jimei University, Xiamen, 361021, China

    Cuihong du, Long Han & Zhijun Xie

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  1. Cuihong du
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  2. Long Han
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  3. Zhijun Xie
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Correspondence to Cuihong du.

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du, C., Han, L. & Xie, Z. Cloning, expression in Escherichia coli, and purification of soluble recombinant duck interleukin-2. World J Microbiol Biotechnol 28, 1495–1501 (2012). https://doi.org/10.1007/s11274-011-0951-8

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  • DOI: https://doi.org/10.1007/s11274-011-0951-8

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