Tabriz University of Medical Sciences Advanced Pharmaceutical Bulletin 2228-5881 16 1 2026 05 19 Optimizing Expression of a Llama-Based Anti-PSMA Nanobody in Escherichia Coli and Its Application in Immunohistochemistry of Prostate Cancer Tissues 62 73 10.34172/apb.025.45447 EN Seyedeh Sheila Seyed-Motahari https://orcid.org/0009-0009-5723-2789 Shahriyar Abdoli Mohammad Ali Shokrgozar Shiva Irani Zahra Sharifzadeh https://orcid.org/0000-0002-4419-0315 Journal Article 10.34172/apb.025.45447 2025 04 11 2025 12 20 Purpose: Nanobodies possess unique properties that make them promising as tumor targeting agents. Prostate-specific membrane antigen (PSMA), overexpressed in prostate cancer, can be an excellent target for prostate cancer diagnosis. This study aimed to express and purify an anti-PSMA nanobody (PSMA-Nb) and assess its potential for detecting PSMA antigen in prostate cancer through immunohistochemistry (IHC). Methods: The PSMA-Nb gene was subcloned into pET-28a and expressed in E. coli Rosetta (DE3) and Rosetta-gami2 under varying IPTG/temperature/time conditions (0.5/1.0/1.5 mM; 16/30/37 °C; 4/16 h). The soluble and inclusion-body fractions were analyzed, and PSMA-Nb was purified via native Ni-NTA, confirmed by SDS-PAGE and anti-c-Myc Western blot. Binding was validated by ELISA against recombinant PSMA and by flow cytometry on LNCaP (PSMA+) and DU145 (PSMA-). For tissue studies, FFPE IHC quantified staining as fractional fluorescent area per mm². Results: PSMA-Nb (~27 kDa) was enriched in E. coli inclusion bodies. A factorial screen identified Rosetta (DE3) with 1 mM IPTG at 37 °C for 16 h as the highest-expressing condition; resulting in a yield of approximately 84 mg/L. ELISA showed dose-dependent binding to recombinant PSMA, and flow cytometry confirmed antigen selectivity (LNCaP 68.5% vs. DU145 2.35%). IHC showed higher PSMA levels in tumor vs. normal tissue with both reagents (PSMA-Nb: 21.61±2.89 vs. 5.82±1.80; commercial antibody: 20.55±3.80 vs. 5.50±2.14; P<0.0001), with no difference between reagents (P=0.9624), supporting analytical validity. Conclusion: The superior features of nanobodies support antibody-based diagnostics in solid tumors. Purified PSMA-Nb detected PSMA on cancer cells and FFPE tissues, indicating a promising tool for prostate cancer diagnosis.