Improved self-cleaving precipitation tags for efficient column free bioseparations

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 s...

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Veröffentlicht in:Protein expression and purification 2024-12, Vol.224, p.106578, Article 106578
Hauptverfasser: Yuan, Hongyu, Prabhala, Sai Vivek, Coolbaugh, Michael J., Stimple, Samuel D., Wood, David W.
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Sprache:eng
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Zusammenfassung: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. •Yeast surface display methodology was used to generate two novel mutants of the Mtu RecA ΔI-CM mini-intein.•The novel mutants ΔI-12 and ΔI-29 follow pH sensitive first order cleaving kinetics.•The novel mutants were fused with the ELP precipitation tag to develop a self-cleaving purification tag method.•These tags resulted in reduced premature cleavage, higher product purities and process yields overall.
ISSN:1046-5928
1096-0279
1096-0279
DOI:10.1016/j.pep.2024.106578