One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand
Cutinase from <i>Fusarium solani pisi</i> is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from <i>E. coil</i&g...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-01-01
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| Series: | Biomimetics |
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| Online Access: | https://www.mdpi.com/2313-7673/9/1/57 |
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| author | Luís P. Fonseca M. Ângela Taipa |
| author_facet | Luís P. Fonseca M. Ângela Taipa |
| author_sort | Luís P. Fonseca |
| collection | DOAJ |
| description | Cutinase from <i>Fusarium solani pisi</i> is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from <i>E. coil</i> extracts is a well-established but time-consuming process, which involves a sequence of two anionic exchange chromatography steps followed by dialysis. Affinity chromatography is the most efficient method for protein purification, the major limitation of its use being often the availability of a ligand selective for a given target protein. Synthetic affinity ligands that specifically recognize certain sites on the surface of proteins are highly desirable for affinity processes due to their cost-effectiveness, durability, and reusability across multiple cycles. Additionally, these ligands establish moderate affinity interactions with the target protein, making it possible to purify proteins under gentle conditions while maintaining high levels of activity recovery. This study aimed to develop a new method for purifying cutinase, utilizing triazine-scaffolded biomimetic affinity ligands. These ligands were previously screened from a biased-combinatorial library to ensure their binding ability to cutinase without compromising its biological function. A lead ligand, designated as 11/3′, [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid], was chosen and directly synthesized onto agarose. Experiments conducted at different scales demonstrated that this ligand (with an affinity constant K<sub>a</sub> ≈ 10<sup>4</sup> M<sup>−1</sup>) exhibited selectivity towards cutinase, enabling the purification of the enzyme from an <i>E. coli</i> crude production medium in a single step. Under optimized conditions, the protein and activity yields reached 25% and 90%, respectively, with a resulting cutinase purity of 85%. |
| first_indexed | 2024-03-08T11:04:12Z |
| format | Article |
| id | doaj.art-42693d8ae4ab456d9df7e6595dfce565 |
| institution | Directory Open Access Journal |
| issn | 2313-7673 |
| language | English |
| last_indexed | 2024-03-08T11:04:12Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomimetics |
| spelling | doaj.art-42693d8ae4ab456d9df7e6595dfce5652024-01-26T15:16:59ZengMDPI AGBiomimetics2313-76732024-01-01915710.3390/biomimetics9010057One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity LigandLuís P. Fonseca0M. Ângela Taipa1Department of Bioengineering, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisbon, PortugalDepartment of Bioengineering, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisbon, PortugalCutinase from <i>Fusarium solani pisi</i> is an enzyme that bridges functional properties between lipases and esterases, with applications in detergents, food processing, and the synthesis of fine chemicals. The purification procedure of recombinant cutinase from <i>E. coil</i> extracts is a well-established but time-consuming process, which involves a sequence of two anionic exchange chromatography steps followed by dialysis. Affinity chromatography is the most efficient method for protein purification, the major limitation of its use being often the availability of a ligand selective for a given target protein. Synthetic affinity ligands that specifically recognize certain sites on the surface of proteins are highly desirable for affinity processes due to their cost-effectiveness, durability, and reusability across multiple cycles. Additionally, these ligands establish moderate affinity interactions with the target protein, making it possible to purify proteins under gentle conditions while maintaining high levels of activity recovery. This study aimed to develop a new method for purifying cutinase, utilizing triazine-scaffolded biomimetic affinity ligands. These ligands were previously screened from a biased-combinatorial library to ensure their binding ability to cutinase without compromising its biological function. A lead ligand, designated as 11/3′, [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid], was chosen and directly synthesized onto agarose. Experiments conducted at different scales demonstrated that this ligand (with an affinity constant K<sub>a</sub> ≈ 10<sup>4</sup> M<sup>−1</sup>) exhibited selectivity towards cutinase, enabling the purification of the enzyme from an <i>E. coli</i> crude production medium in a single step. Under optimized conditions, the protein and activity yields reached 25% and 90%, respectively, with a resulting cutinase purity of 85%.https://www.mdpi.com/2313-7673/9/1/57recombinant cutinase<i>E. coli</i>affinity chromatographybiomimetic affinity ligandstriazine-scaffoldcombinatorial library |
| spellingShingle | Luís P. Fonseca M. Ângela Taipa One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand Biomimetics recombinant cutinase <i>E. coli</i> affinity chromatography biomimetic affinity ligands triazine-scaffold combinatorial library |
| title | One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand |
| title_full | One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand |
| title_fullStr | One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand |
| title_full_unstemmed | One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand |
| title_short | One-Step Purification of Recombinant Cutinase from an <i>E. coli</i> Extract Using a Stabilizing Triazine-Scaffolded Synthetic Affinity Ligand |
| title_sort | one step purification of recombinant cutinase from an i e coli i extract using a stabilizing triazine scaffolded synthetic affinity ligand |
| topic | recombinant cutinase <i>E. coli</i> affinity chromatography biomimetic affinity ligands triazine-scaffold combinatorial library |
| url | https://www.mdpi.com/2313-7673/9/1/57 |
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