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Article

Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris

  • Authors:
    • Manman Su
    • Weiqin Chang
    • Kun Zhang
    • Manhua Cui
    • Shuying Wu
    • Tianmin Xu
  • View Affiliations / Copyright

    Affiliations: Department of Obstetrics and Gynecology, The Second Hospital, Jilin University, Changchun, Jilin 130041, P.R. China
  • Pages: 1179-1185
    |
    Published online on: November 25, 2015
       https://doi.org/10.3892/or.2015.4448
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Abstract

Melittin is well known to possess cytolytic activity with wide-spectrum lytic properties and its potential use as an agent to treat several types of cancer has been tested. Due to the non-specific toxicity, melittin can impair not only cancer cells but also normal tissue. Thus, tumor-targeted toxins may be helpful for developing novel anticancer therapeutics. The urokinase-type plasminogen activator (uPA) plays a central role in tissue remodelling events occurring in normal physiology and in pathophysiology, including cancer invasion and metastasis. Heartening findings showed that uPA receptor is predominantly expressed on many types of cancer. Therefore, the amino-terminal fragment (ATF) of uPA which was able to identify and bond with cancer cells was used as the cell-targeting domain to make up tumor-targeted toxin in this study. In the present study, pPICZαC-ATF-melittin eukaryotic expression vector was successfully constructed. After transformed into P. pastoris and induced by methanol, rATF-mellitin was detected by SDS-PAGE and western blot analysis. After induction with methanol, the expression level of rATF-mellitin was 312 mg/l in 80-l fermentor. rATF‑mellitin was purified to >95% purity using SP Sepharose ion exchange chromatography and source™ 30 RPC with 67.2% recovery. Cell proliferation assay showed that rATF-melittin inhibited growth of SKOV3 cells and had no cytotoxicity effect on normal cells. For the first time, we established a stable and effective rATF-mellitin P. pastoris expression system to obtain a high level of expression of secreted rATF-mellitin which was purified by a highly efficient purification procedure.
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Copy and paste a formatted citation
Spandidos Publications style
Su M, Chang W, Zhang K, Cui M, Wu S and Xu T: Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris. Oncol Rep 35: 1179-1185, 2016.
APA
Su, M., Chang, W., Zhang, K., Cui, M., Wu, S., & Xu, T. (2016). Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris. Oncology Reports, 35, 1179-1185. https://doi.org/10.3892/or.2015.4448
MLA
Su, M., Chang, W., Zhang, K., Cui, M., Wu, S., Xu, T."Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris". Oncology Reports 35.2 (2016): 1179-1185.
Chicago
Su, M., Chang, W., Zhang, K., Cui, M., Wu, S., Xu, T."Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris". Oncology Reports 35, no. 2 (2016): 1179-1185. https://doi.org/10.3892/or.2015.4448
Copy and paste a formatted citation
x
Spandidos Publications style
Su M, Chang W, Zhang K, Cui M, Wu S and Xu T: Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris. Oncol Rep 35: 1179-1185, 2016.
APA
Su, M., Chang, W., Zhang, K., Cui, M., Wu, S., & Xu, T. (2016). Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris. Oncology Reports, 35, 1179-1185. https://doi.org/10.3892/or.2015.4448
MLA
Su, M., Chang, W., Zhang, K., Cui, M., Wu, S., Xu, T."Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris". Oncology Reports 35.2 (2016): 1179-1185.
Chicago
Su, M., Chang, W., Zhang, K., Cui, M., Wu, S., Xu, T."Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris". Oncology Reports 35, no. 2 (2016): 1179-1185. https://doi.org/10.3892/or.2015.4448
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