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Research in Pharmaceutical Sciences، جلد ۲۰، شماره ۳، صفحات ۳۷۳-۳۹۱
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عنوان انگلیسی Expression and functional characterization of an anti-CD22 scFv targeting B-cell malignancies
چکیده انگلیسی مقاله Background and purpose: Single-chain variable fragments (scFvs) offer advantages over full-length monoclonal antibodies in cancer therapy, including reduced size, lower production costs, and easier handling. However, Escherichia coli ( E. coli ) often leads to the formation and aggregation of inclusion bodies (IBs). This study aimed to optimize the expression and purification of an anti-CD22 scFv (CD22-scFv) in E. coli and evaluate its functional properties. Experimental approach: The CD22-scFv construct was subcloned into pET-28a(+) and expressed in E. coli strains Rosetta (DE3) and Rosetta-gami 2. To overcome IBs formation, two purification methods were employed to enhance soluble protein production: hybrid conditions, a novel one-step immobilized metal affinity chromatography (IMAC)-based on-column refolding method was employed, using gradually decreasing urea and increasing imidazole concentrations; native conditions, expression parameters (IPTG concentration, post-induction temperature, and time) were optimized, followed by IMAC. The CD22-scFv binding to CD22 antigen and its anti-proliferative effects on target cells were assessed via flow cytometry and MTT assay. Findings/Results: CD22-scFv was successfully expressed in Rosetta (DE3) but not Rosetta-gami 2. Hybrid purification yielded 15.86 mg/L protein, outperforming native purification (3.65 mg/L). Flow cytometry confirmed the binding of native- and hybrid-purified CD22-scFv to CD22 Raji cells with 75.5% and 55.8% efficiency, respectively. Native-purified CD22-scFv significantly inhibited Raji cell proliferation while sparing CD22 − cells. Conclusion and implications: This study established a scalable and cost-effective strategy for producing functional CD22-scFv with high specificity and anti-proliferative effects. The findings highlight its potential for targeted therapies and diagnostics, warranting further in vivo and clinical studies.    
کلیدواژه‌های انگلیسی مقاله CD22-scFv,E. coli expression system,IMAC purification,Immunotherapy,On-column refolding,Protein solubility.
نویسندگان مقاله | Monireh Gholizadeh
School of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran.


| Shahriyar Abdoli
Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.


| Shafieeh Mansoori
Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran.


| Arash Arashkia
Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.


| Farhad Riazi-Rad
Pediatric Cell and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran, Iran.


| Amir Ali Hamidieh
Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.


| Mohammad Nouri
Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.


| Zahra Sharifzadeh


نشانی اینترنتی http://rps.mui.ac.ir/index.php/jrps/article/view/2320
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زبان مقاله منتشر شده en
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نوع مقاله منتشر شده Original Article
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