Scientific Reports (Mar 2025)
A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli
- James Bosco,
- Emily Gagliano,
- Kassandra L. Boshae,
- John P. Statz,
- Timothy B. Wheeler,
- DeAnna Cuello,
- Ashlyn Sliter,
- Christian Newby,
- Bernice Lin,
- Aysha Demeler,
- C. Logan Pierpont,
- Cindee Yates-Hansen,
- Matthew J. Sydor,
- Maria E. Ferrini,
- Kellie C. Kuch,
- Brandon S. Cooper,
- Beverly J. Piggott,
- Sarah J. Certel,
- Kasper B. Hansen,
- Stephen R. Sprang,
- Bruce Bowler,
- Levi McClelland,
- Mehmet Berkmen,
- Ekaterina Voronina
Affiliations
- James Bosco
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Emily Gagliano
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Kassandra L. Boshae
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- John P. Statz
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Timothy B. Wheeler
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- DeAnna Cuello
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Ashlyn Sliter
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Christian Newby
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Bernice Lin
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Aysha Demeler
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge
- C. Logan Pierpont
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Cindee Yates-Hansen
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Matthew J. Sydor
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Maria E. Ferrini
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Kellie C. Kuch
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Brandon S. Cooper
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Beverly J. Piggott
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Sarah J. Certel
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Kasper B. Hansen
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Stephen R. Sprang
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Bruce Bowler
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Levi McClelland
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- Mehmet Berkmen
- New England Biolabs
- Ekaterina Voronina
- Division of Biological Sciences, Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, Center for Structural and Functional Neuroscience, Center for Environmental Health Sciences, University of Montana
- DOI
- https://doi.org/10.1038/s41598-025-91954-5
- Journal volume & issue
-
Vol. 15,
no. 1
pp. 1 – 18
Abstract
Abstract Protein production using Escherichia coli is a cornerstone of modern biotechnology. In this study, we developed a novel auto-expression medium to maximize protein production. Each E. coli strain tested was capable of auto-expression in response to galactose, including strains in which the endogenous lacZ had been disrupted. This provides key evidence that galactose can regulate the lac operon independent of known lac operon-regulated metabolism. The enhanced capabilities of the novel auto-expression medium were documented across protein production systems including (1) increased yields for routinely expressed proteins (e.g. eGFP), (2) improved expression of human cytochrome c within a dual expression system, (3) robust auto-expression in lacZ-deficient strains producing proteins with challenging disulfide bonds, and (4) reproducible 8-fold increase in SpCas9 yields, at ≥ 95% purity. This novel medium can streamline production and improve yields for routine as well as challenging proteins, accelerating recombinant protein production and creating new opportunities in biotechnology and structural biology.
