Iraninan Biology Society Journal of Cellular and Molecular Research (Iranian Journal of Biology) 2383-2738 37 4 2025 02 19 Design and production of a new bioluminescent probe for molecular detection of Staphylococcus aureus Design and production of a new bioluminescent probe for molecular detection of Staphylococcus aureus 320 331 2248 FA Yasaman Mazidi Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, I.R. of Iran Rahman Emamzadeh Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, I.R. of Iran 0000-0002-7924-1135 Mahboobeh Nazari Endocrine Research Center, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, I.R. of Iran Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, I.R. of Iran S.shahrbanoo Jafari Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, I.R. of Iran Journal Article 2022 05 24 Staphylococcus aureus is an important pathogen in humans and animals. This bacterium is important due to its great ability to gain resistance to a variety of antibiotics and cause severe diseases. Identifying acute infections caused by this bacterium using conventional methods such as culture is costly and time-consuming.In this study, the design and development of a new probe that specifically detects S. aureus by emitting a luminescence signal is carried out. The new recombinant probe, named Rluc-(ZpA963) 2, has the gene sequence encoding Renilla luciferase (Rluc) in fusion with the gene sequence encoding affibody (ZpA963)2. Protein expression was performed in Escherichia coli BL21 and protein purification was carried out by affinity chromatography. Kinetics studies show that Km of Rluc in the new probe is similar to recombinant Rluc; and are 1.07 µM and 1.22 µM, respectively. Therefore, the affinity for binding to cholentrazine does not change in Rluc-(ZpA963) 2. The binding of Rluc-(ZpA963)2 to S. aureus also show that the new probe is specifically capable of binding to bacteria at 4° C and 25 ° C. Moreover, the strongest signal is recorded in S. aureus growing in the Brain Heart broth. The present study may be used to develop commercial kits in the future. Staphylococcus aureus is an important pathogen in humans and animals. This bacterium is important due to its great ability to gain resistance to a variety of antibiotics and cause severe diseases. Identifying acute infections caused by this bacterium using conventional methods such as culture is costly and time-consuming.In this study, the design and development of a new probe that specifically detects S. aureus by emitting a luminescence signal is carried out. The new recombinant probe, named Rluc-(ZpA963) 2, has the gene sequence encoding Renilla luciferase (Rluc) in fusion with the gene sequence encoding affibody (ZpA963)2. Protein expression was performed in Escherichia coli BL21 and protein purification was carried out by affinity chromatography. Kinetics studies show that Km of Rluc in the new probe is similar to recombinant Rluc; and are 1.07 µM and 1.22 µM, respectively. Therefore, the affinity for binding to cholentrazine does not change in Rluc-(ZpA963) 2. The binding of Rluc-(ZpA963)2 to S. aureus also show that the new probe is specifically capable of binding to bacteria at 4° C and 25 ° C. Moreover, the strongest signal is recorded in S. aureus growing in the Brain Heart broth. The present study may be used to develop commercial kits in the future. https://cell.ijbio.ir/article_2248_be6c7b094f88532b6c6b35bbcd525ee8.pdf