Design of a bacterial system for the production and purification of recombinant NcGRA7 protein using Staphylococcus aureus Sortase A Activity

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Published: Feb 12, 2026
DOI: https://doi.org/10.33262/anatomiadigital.v9i1.2.3605
Keywords:
Sortase A, recombinant proteins, protein purification, NcGRA7, immobilized metal affinity chromatography.

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Janneth Fernanda Cárdenas Cordero
Universidad de Cuenca (UCUENCA)
Antonio Javier Vallecillo Maza
Universidad de Cuenca (UCUENCA)

Abstract

Introduction. Sortase A (SrtA) is a cysteine transpeptidase found in Gram-positive bacteria, where it catalyzes site-specific peptide bond cleavage and ligation. Due to these properties, SrtA widely applied as a biotechnological tool for the production and purification of recombinant proteins, allowing controlled proteolytic processing and improved purification efficiency. Objective. This study aimed to develop a bacterial system for the production and purification of recombinant proteins based on the enzymatic activity of Staphylococcus aureus Sortase A (Sa-SrtA). Methodology. A modified pET15b NT-Histidine vector (Novagen) was constructed by incorporating the coding sequence of Δ60Sa-SrtA (GenBank Acc. No.: AF162687) from S. aureus (ATCC 12598), followed by the consensus recognition motif LPET4G. The antigenic protein NcGRA7 from Neospora caninum (GenBank Acc. No.: U82229) was cloned downstream of this motif, generating the fusion construct His-tag–Δ60Sa-SrtA–LPET4G–NcGRA7. Protein expression was conducted in Escherichia coli Rosetta 2 (DE3), and purification was performed by immobilized metal affinity chromatography (IMAC) using Ni-NTA resin. Enzymatic cleavage was induced in the presence of Ca²⁺ and triglycine, enabling the release of the target protein. Results. A bacterial model was obtained for the generation of purified recombinant proteins by the enzymatic activity of Δ60Sa-SrtA. The system allowed the purification of the recombinant protein of N. caninum 4G-NcGRA7, that could be used for the generation of the diagnostic system. Conclusion. The Δ60Sa-SrtA-based system represents an effective and reliable strategy for the production and purification of recombinant proteins, with potential applications in diagnostic and biotechnological developments. General Area of Study: Biology. Specific area of study: Molecular Biology. Type of study: Original articles.

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How to Cite
Cárdenas Cordero, J. F., & Vallecillo Maza, A. J. (2026). Design of a bacterial system for the production and purification of recombinant NcGRA7 protein using Staphylococcus aureus Sortase A Activity. Anatomía Digital, 9(1.2), 66-82. https://doi.org/10.33262/anatomiadigital.v9i1.2.3605
Issue
Vol. 9 No. 1.2 (2026): Medicina Metabólica
Section
Articulos de revisión bibliográfica
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