Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic Heating
ABSTRACT Komagataella phaffii is among the most widely used expression systems, with methanol‐inducible promoters being preferred for protein expression due to their stringent regulation and exceptional strength. However, the applicability of this system, particularly in food and pharmaceutical prod...
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| Format: | Article |
| Language: | English |
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Wiley
2025-06-01
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| Series: | Microbial Biotechnology |
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| Online Access: | https://doi.org/10.1111/1751-7915.70183 |
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| author | Ibrahim Dagci Seyda Yildiz Arslan Kubra Solak Melek Acar Yagmur Unver Ahmet Mavi |
| author_facet | Ibrahim Dagci Seyda Yildiz Arslan Kubra Solak Melek Acar Yagmur Unver Ahmet Mavi |
| author_sort | Ibrahim Dagci |
| collection | DOAJ |
| description | ABSTRACT Komagataella phaffii is among the most widely used expression systems, with methanol‐inducible promoters being preferred for protein expression due to their stringent regulation and exceptional strength. However, the applicability of this system, particularly in food and pharmaceutical products, is limited by methanol's toxic and pro‐inflammatory properties. Therefore, obtaining a novel methanol‐free expression system is necessary. In this study, we obtained a novel expression plasmid, pHSPαA, carrying the HSP70 promoter (PHSP70) to regulate heterologous expression through heat induction. The extracellular expression of azurin was achieved using this methanol‐free system under the control of PHSP70, induced by either magnetic or non‐magnetic heating. To enhance heat‐induced expression, recombinant cells were immobilised with Fe3O4@PEI25 kDa nanoparticles, which facilitated heat release under an AC magnetic field, thereby increasing cell permeability and protein secretion. A time‐dependent increase in protein expression was observed in non‐magnetic heating but not under magnetic heating. However, immobilised cells exhibited a higher protein secretion capacity compared to non‐immobilised cells. These findings suggest that the novel methanol‐free expression system represents a promising alternative for heterologous gene expression, particularly for the production of therapeutically relevant and food‐grade recombinant proteins. |
| format | Article |
| id | doaj-art-e5eba15ea49c4817a8b861e35377f8d1 |
| institution | DOAJ |
| issn | 1751-7915 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj-art-e5eba15ea49c4817a8b861e35377f8d12025-08-20T03:24:02ZengWileyMicrobial Biotechnology1751-79152025-06-01186n/an/a10.1111/1751-7915.70183Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic HeatingIbrahim Dagci0Seyda Yildiz Arslan1Kubra Solak2Melek Acar3Yagmur Unver4Ahmet Mavi5Department of Molecular Biology and Genetics Institute of Science and Technology, Atatürk University Erzurum TürkiyeDepartment of Molecular Biology and Genetics Institute of Science and Technology, Atatürk University Erzurum TürkiyeDepartment of Molecular Biology and Genetics Faculty of Science, Atatürk University Erzurum TürkiyeDepartment of Molecular Biology and Genetics Institute of Science and Technology, Atatürk University Erzurum TürkiyeDepartment of Molecular Biology and Genetics Faculty of Science, Atatürk University Erzurum TürkiyeEast Anatolia High Technology Application and Research Center (DAYTAM) Atatürk University Erzurum TürkiyeABSTRACT Komagataella phaffii is among the most widely used expression systems, with methanol‐inducible promoters being preferred for protein expression due to their stringent regulation and exceptional strength. However, the applicability of this system, particularly in food and pharmaceutical products, is limited by methanol's toxic and pro‐inflammatory properties. Therefore, obtaining a novel methanol‐free expression system is necessary. In this study, we obtained a novel expression plasmid, pHSPαA, carrying the HSP70 promoter (PHSP70) to regulate heterologous expression through heat induction. The extracellular expression of azurin was achieved using this methanol‐free system under the control of PHSP70, induced by either magnetic or non‐magnetic heating. To enhance heat‐induced expression, recombinant cells were immobilised with Fe3O4@PEI25 kDa nanoparticles, which facilitated heat release under an AC magnetic field, thereby increasing cell permeability and protein secretion. A time‐dependent increase in protein expression was observed in non‐magnetic heating but not under magnetic heating. However, immobilised cells exhibited a higher protein secretion capacity compared to non‐immobilised cells. These findings suggest that the novel methanol‐free expression system represents a promising alternative for heterologous gene expression, particularly for the production of therapeutically relevant and food‐grade recombinant proteins.https://doi.org/10.1111/1751-7915.70183alternating magnetic fieldHSP70 promoterKomagataella phaffiimagnetic nanoparticlesmethanol‐free expression |
| spellingShingle | Ibrahim Dagci Seyda Yildiz Arslan Kubra Solak Melek Acar Yagmur Unver Ahmet Mavi Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic Heating Microbial Biotechnology alternating magnetic field HSP70 promoter Komagataella phaffii magnetic nanoparticles methanol‐free expression |
| title | Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic Heating |
| title_full | Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic Heating |
| title_fullStr | Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic Heating |
| title_full_unstemmed | Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic Heating |
| title_short | Methanol‐Free Protein Expression in Komagataella phaffii With Magnetic or Non‐Magnetic Heating |
| title_sort | methanol free protein expression in komagataella phaffii with magnetic or non magnetic heating |
| topic | alternating magnetic field HSP70 promoter Komagataella phaffii magnetic nanoparticles methanol‐free expression |
| url | https://doi.org/10.1111/1751-7915.70183 |
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