Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation |
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| Affiliation: | 1. Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia;2. University of Latvia, Raina bulv. 19, Riga LV-1586, Latvia;3. Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia;1. Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China;2. Department of Clinical Pharmacology, General Hospital of Beijing Military Command of PLA, Beijing 100700, PR China;1. Department of Chemistry, School of Life Sciences, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei 230027, China;2. High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, China;1. Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan;2. Department of Botany and Microbiology, Faculty of Science, Minia University, Minia 61519, Egypt;1. Bioorganic Chemistry, Gebäude NWI, University of Bayreuth, 95440 Bayreuth, Germany;2. Department of Chemistry, Graduate School of Science, Osaka University, 1-1, Machikaneyama, Toyonaka, Osaka 560-0043, Japan |
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| Abstract: | Staphylococcus aureus sortase A is a transpeptidase that is extensively used in various protein research applications. Sortase A is highly selective and does not require any cofactors for the catalysis of protein ligation and, importantly, can be produced in high yields. However, the primary disadvantage of this transpeptidase is its inability to access the recognition site within the highly structured regions of folded substrates. To overcome this problem, we developed an Escherichia coli expression system that produces milligram quantities of circularly closed sortase A; efficient enzyme cyclization was achieved by Synechocystis sp. PCC6803 intein-mediated post-translational splicing. The structural integrity of circular sortase A and its biochemical characteristics were compared to those of the linear enzyme analog and were found to be similar under native conditions. Additionally, the modified sortase was active at concentrations of urea up to 3 M and was capable of efficient catalytic protein–protein coupling, as shown by the ligation of purified glutathione-S-transferase and green fluorescence protein. In contrast to the circular enzyme, linear sortase A was unable to mediate the ligation of substrate proteins under the same conditions. Therefore, the proposed circular sortase A has improved enzymatic properties and has applications in advanced protein engineering and design. |
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| Keywords: | Denaturing conditions Enzyme stability Intein-mediated cyclization Protein ligation Sortase A |
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