Improved self-cleaving precipitation tags for efficient column free bioseparations.
Autor: | Yuan H; William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, 43210, USA., Prabhala SV; William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, 43210, USA., Coolbaugh MJ; William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, 43210, USA., Stimple SD; William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, 43210, USA., Wood DW; William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH, 43210, USA. Electronic address: wood.750@osu.edu. |
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Jazyk: | angličtina |
Zdroj: | Protein expression and purification [Protein Expr Purif] 2024 Dec; Vol. 224, pp. 106578. Date of Electronic Publication: 2024 Aug 15. |
DOI: | 10.1016/j.pep.2024.106578 |
Abstrakt: | Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale. (Copyright © 2024 Elsevier Inc. All rights reserved.) |
Databáze: | MEDLINE |
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