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Expression and Purification of Bioactive High-Purity Recombinant Mouse SPP1 in Escherichia coli

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Abstract

Secreted phosphoprotein 1 (SPP1) is a phosphorylated acidic glycoprotein. It is broadly expressed in a variety of tissues, and it is involved in a number of physiological and pathological events, including cancer metastasis, tissues remodeling, pro-inflammation regulation, and cell survival. SPP1 has shown its function of protecting tissues and organs against injury and wound, giving itself potentials to become a therapy target or giving its antibodies of other counter-acting reagents potentials to become drug candidates. Non-tagged (native) recombinant SPP1 would be valuable in therapeutic and pharmaceutical researches. In our study, mouse Spp1 DNA fragment without signal peptide was built in pET28a(+) vector and transformed into Escherichia coli BL21 (DE3). The recombinant mouse SPP1 (rmSPP1) was then expressed in bacteria upon induction by isopropyl β-d-thiogalactopyranoside (IPTG). The abundance of rmSPP1 was increased using isoelectric precipitation and ammonium sulfate fractionation methods, and anion and cation exchange chromatography was employed to further purify rmSPP1. Finally, we got rmSPP1 product with 12.8 % productivity, 97 % purity, satisfactory bioactivity, and low endotoxin content.

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Acknowledgments

We thank Ruliang Zhang from General Regeneratives Limited Company (Shanghai, China) for analyzing protein purity and Kathleen L. Lengel from Pennsylvania State University (Hershey, PA) for reviewing our manuscript. This work was supported by the National Natural and Science Foundation of China (81273573; 81302825) and Key Laboratory of Urban Agriculture (South) Ministry of Agriculture (10UA002).

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Authors and Affiliations

  1. Shanghai Key Laboratory of Veterinary Biotechnology, College of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd, 200240, Shanghai, People’s Republic of China

    Yunsheng Yuan, Xiyuan Zhang, Shunyan Weng, Wen Guan & Yan Yu

  2. Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, College of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Rd, 200240, Shanghai, People’s Republic of China

    Yunsheng Yuan & Yan Yu

  3. Department of Tumor Biology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, E407 New Research Building, 3970 Reservoir Road NW, Washington, DC, 20057, USA

    Xiyuan Zhang

  4. Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA, 94609, USA

    Di Xiang

  5. Laboratory of Regeneromics, School of Pharmacy, Shanghai Jiaotong University, 800 Dongchuan Rd, 200240, Shanghai, People’s Republic of China

    Jin Gao, Jingjing Li & Wei Han

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  1. Yunsheng Yuan
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  2. Xiyuan Zhang
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  3. Shunyan Weng
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  4. Wen Guan
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  5. Di Xiang
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  6. Jin Gao
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  7. Jingjing Li
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  8. Wei Han
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  9. Yan Yu
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Correspondence to Yan Yu.

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Figure 1

Optimization of rmSpp1 expression condition in bacterial. A, When the OD600 of medium reached 1.0, indicated concentration of IPTG was added into the medium to induce rmSPP1expression in bacteria. Bacteria would be harvested at 6 hours post IPTG, and level of rmSpp1 was analyzed by 15% SDS-PAGE. B, Bacteria were harvested at indicated time points after 1mM of IPTG being added to medium. Cells lysate was analyzed by 15% SDS-PAGE. (JPEG 39 kb)

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Yuan, Y., Zhang, X., Weng, S. et al. Expression and Purification of Bioactive High-Purity Recombinant Mouse SPP1 in Escherichia coli . Appl Biochem Biotechnol 173, 421–432 (2014). https://doi.org/10.1007/s12010-014-0849-7

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  • DOI: https://doi.org/10.1007/s12010-014-0849-7

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