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; De Anna 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

doi:10.1038/s41598-025-91954-5

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, De Anna 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. SprangBruce Bowler, Levi McClelland, Mehmet Berkmen, Ekaterina Voronina

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Article number	8936
Journal	Scientific Reports
Volume	15
Issue number	1
DOIs	https://doi.org/10.1038/s41598-025-91954-5
State	Published - Mar 15 2025

Keywords

Cytochromes c/metabolism
Escherichia coli/genetics
Galactose/metabolism
Gene Expression Regulation, Bacterial
Humans
Lac Operon/genetics
Recombinant Proteins/metabolism

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Bosco, J., Gagliano, E., Boshae, K. L., Statz, J. P., Wheeler, T. B., Cuello, D. A., Sliter, A., Newby, C., Lin, B., Demeler, A., Pierpont, C. L., Yates-Hansen, C., Sydor, M. J., Ferrini, M. E., Kuch, K. C., Cooper, B. S., Piggott, B. J., Certel, S. J., Hansen, K. B., ... Voronina, E. (2025). A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli. Scientific Reports, 15(1), Article 8936. https://doi.org/10.1038/s41598-025-91954-5

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title = "A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli",

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.",

keywords = "Cytochromes c/metabolism, Escherichia coli/genetics, Galactose/metabolism, Gene Expression Regulation, Bacterial, Humans, Lac Operon/genetics, Recombinant Proteins/metabolism",

author = "James Bosco and Emily Gagliano and Boshae, {Kassandra L.} and Statz, {John P.} and Wheeler, {Timothy B.} and Cuello, {De Anna} and Ashlyn Sliter and Christian Newby and Bernice Lin and Aysha Demeler and Pierpont, {C. Logan} and Cindee Yates-Hansen and Sydor, {Matthew J.} and Ferrini, {Maria E.} and Kuch, {Kellie C.} and Cooper, {Brandon S.} and Piggott, {Beverly J.} and Certel, {Sarah J.} and Hansen, {Kasper B.} and Sprang, {Stephen R.} and Bruce Bowler and Levi McClelland and Mehmet Berkmen and Ekaterina Voronina",

note = "{\textcopyright} 2025. The Author(s).",

year = "2025",

month = mar,

day = "15",

doi = "10.1038/s41598-025-91954-5",

language = "English",

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issn = "2045-2322",

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Bosco, J, Gagliano, E, Boshae, KL, Statz, JP, Wheeler, TB, Cuello, DA, Sliter, A, Newby, C, Lin, B, Demeler, A, Pierpont, CL, Yates-Hansen, C, Sydor, MJ, Ferrini, ME, Kuch, KC, Cooper, BS , Piggott, BJ , Certel, SJ , Hansen, KB , Sprang, SR , Bowler, B , McClelland, L, Berkmen, M & Voronina, E 2025, 'A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli', Scientific Reports, vol. 15, no. 1, 8936. https://doi.org/10.1038/s41598-025-91954-5

TY - JOUR

T1 - A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli

AU - Bosco, James

AU - Gagliano, Emily

AU - Boshae, Kassandra L.

AU - Statz, John P.

AU - Wheeler, Timothy B.

AU - Cuello, De Anna

AU - Sliter, Ashlyn

AU - Newby, Christian

AU - Lin, Bernice

AU - Demeler, Aysha

AU - Pierpont, C. Logan

AU - Yates-Hansen, Cindee

AU - Sydor, Matthew J.

AU - Ferrini, Maria E.

AU - Kuch, Kellie C.

AU - Cooper, Brandon S.

AU - Piggott, Beverly J.

AU - Certel, Sarah J.

AU - Hansen, Kasper B.

AU - Sprang, Stephen R.

AU - Bowler, Bruce

AU - McClelland, Levi

AU - Berkmen, Mehmet

AU - Voronina, Ekaterina

PY - 2025/3/15

Y1 - 2025/3/15

N2 - 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.

AB - 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.

KW - Cytochromes c/metabolism

KW - Escherichia coli/genetics

KW - Galactose/metabolism

KW - Gene Expression Regulation, Bacterial

KW - Humans

KW - Lac Operon/genetics

KW - Recombinant Proteins/metabolism

UR - http://www.scopus.com/inward/record.url?scp=105000198505&partnerID=8YFLogxK

U2 - 10.1038/s41598-025-91954-5

DO - 10.1038/s41598-025-91954-5

M3 - Article

C2 - 40089537

AN - SCOPUS:105000198505

SN - 2045-2322

VL - 15

JO - Scientific Reports

JF - Scientific Reports

IS - 1

M1 - 8936

ER -

A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli

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Keywords

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