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Expression, Purification, and Characterization of Recombinant Protein GX1-rmhTNFα

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Abstract

A phage-displayed peptide CGNSNPKSC (GX1) was obtained previously in our lab, which could specifically bind to the vasculature of human gastric cancer. GX1-rmhTNFα was a fusion protein of GX1 and recombinant mutant human tumor necrosis factor α (rmhTNFα), which was designed by us with the expectation of enhancing selectivity of rmhTNFα. The DNA fragment encoding GX1 was cloned into the vector pBV220 with rmhTNFα between the EcoRI site and the BamHI site, and then expressed in Escherichia coli DH5α by temperature induction. Subsequently, E. coli DH5α was lysed, and the GX1-rmhTNFα protein was found in both soluble form and inclusion bodies. The protein was fractionated with ammonium sulfate deposition from 30% to 60%, and purified by cation and anion exchange chromatography using SP Sepharose Fast Flow column and Q Sepharose Fast Flow column. The purity of protein was then identified by SDS-PAGE and HPLC. Subsequent studies showed that GX1-rmhTNFα had high bioactivity of 5.65 × 108 IU/ml, which was similar with natural human TNFα and could reach the tumor site relatively faster than rmhTNFα.

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Abbreviations

HPLC:

High performance liquid chromatography

FMMU:

The fourth military medical university

ELISA:

Enzyme-linked immunosorbent assay

PBST:

Phosphate buffer solution tween-20

HRP:

Horse radish peroxidase

SPECT:

Single photon emission computed tomography

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Acknowledgments

We thank Yingqi Zhang (Professor, Biotechnology Center of FMMU, China) for the new recombinant human TNFα. This study was supported by the National High-Tech Project of China 2006AA02Z103, 2006AA02A402; the Key Project of PLA 06G087; the National Key Project of Basic Research 2004CB518702.

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Authors and Affiliations

  1. State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, The Fourth Military Medical University, Xi’an, 710032, Shaanxi Province, People’s Republic of China

    Shanshan Cao, Xiaohua Li, Jun Wang, Wenqi Du, Yu Han, Haifeng Jin, Lina Zhao, Kaichun Wu & Daiming Fan

  2. State Key Laboratory of Cancer Biology and Biotechnology Center, The Fourth Military Medical University, Xi’an, Shaanxi, China

    Yan Liu & Yingqi Zhang

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  1. Shanshan Cao
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  2. Yan Liu
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Correspondence to Kaichun Wu.

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Shanshan Cao and Yan Liu contribute equally to this manuscript.

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Cao, S., Liu, Y., Li, X. et al. Expression, Purification, and Characterization of Recombinant Protein GX1-rmhTNFα. Mol Biotechnol 43, 1–7 (2009). https://doi.org/10.1007/s12033-009-9170-z

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