Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate‑specific membrane antigen

Tao,Rong; Ni,Zhenhua; Liu,Chong; Zhu,Min; Ji,Xiaowen; Chen,Xuemin; Shen,Jiangfan; Tu,Shaohua

doi:10.3892/etm.2016.3005

March-2016 Volume 11 Issue 3

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March-2016 Volume 11 Issue 3

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Article Open Access

Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate‑specific membrane antigen

Authors:
- Rong Tao
- Zhenhua Ni
- Chong Liu
- Min Zhu
- Xiaowen Ji
- Xuemin Chen
- Jiangfan Shen
- Shaohua Tu
View Affiliations / Copyright

Affiliations: Department of Nuclear Medicine, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, P.R. China

Copyright: © Tao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Pages: 747-752
|
Published online on: January 18, 2016

https://doi.org/10.3892/etm.2016.3005
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Abstract

The present study aimed to identify, express and purify an immunogenic fragment in the ectodomain of prostate‑specific membrane antigen (PSMA) within a fusion protein. The PSMA amino acid sequence published in National Center for Biotechnology Information GenBank was used to determine sequence homology and immunogenic index analyses, additionally using BLASTN, Protean and ExPASy software to predict the polypeptide sequences of immunogenic epitopes. The gene sequence encoding the ectodomain of the polypeptide immunogenic fragments, containing the identified immunogenic epitopes, was generated using whole‑gene synthesis. Prokaryotic expression vector pET-32a-r-ectodomain‑PSMA was constructed and the recombinant plasmids were transformed into competent BL21 (DE3) Escherichia coli, which was followed by induction of recombinant protein expression using isopropyl‑β‑D‑thiogalactopyranoside. Fusion proteins were isolated and purified using affinity chromatography and their immune activity was subsequently investigated using western blot analysis. Purified protein was used to immunize BALB/c mice in order to generate polyclonal antibodies, and the binding of polyclonal antibodies to prostate cancer cell lines in vitro was evaluated using flow cytometry. A total of 3 polypeptide fragments with high specificity were identified following analysis using numerous software packages, and the gene sequences encoding regions containing the 2 most immunogenic fragments were synthesized and successfully inserted into the prokaryotic expression vector pET‑32a‑r‑ectodomain‑PSMA. The recombinant PSMA protein fragment had a molecular weight of ~50 kDa and 95% purity. Western blot analysis revealed that the r‑ectodomain‑PSMA fusion protein specifically bound to the anti‑PSMA ectodomain monoclonal antibody. Flow cytometry demonstrated that polyclonal antibodies raised against these recombinant proteins could specifically bind to PSMA‑positive LNCaP cells, but not to PSMA‑negative PC‑3 cells. An immunogenic fragment in the ectodomain of PSMA was successfully expressed and purified. The present study, therefore, provides a basis for the preparation of an anti‑PSMA small humanized monoclonal antibody.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate‑specific membrane antigen

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