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Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.

Publication ,  Journal Article
Menacho-Melgar, R; Lynch, MD
Published in: Bio-protocol
January 2022

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly, protein extraction methods are predominantly restricted to chemical methods, and mechanical methods reliant on expensive specialized equipment more tuned for large-scale applications. In this article, we outline detailed protocols for the use of an engineered autolysis/autohydrolysis E. coli strain, in two-stage fermentations in shake-flasks. This two-stage fermentation protocol does not require optimization of expression conditions and results in high protein titers. Cell lysis in an engineered strain is tightly controlled and only triggered post-culture by addition of a 0.1% detergent solution. Upon cell lysis, a nuclease digests contaminating host oligonucleotides, which facilitates sample handling. This method has been validated for use at different scales, from microtiter plates to instrumented bioreactors. Graphic abstract: Two-stage protein expression, cell autolysis and DNA/RNA autohydrolysis. Reprinted with permission from Menacho-Melgar et al. (2020a). Copyright 2020 John Wiley and Sons.

Duke Scholars

Published In

Bio-protocol

DOI

EISSN

2331-8325

ISSN

2331-8325

Publication Date

January 2022

Volume

12

Issue

2

Start / End Page

e4297

Related Subject Headings

  • 32 Biomedical and clinical sciences
  • 31 Biological sciences
 

Citation

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Menacho-Melgar, R., & Lynch, M. D. (2022). Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli. Bio-Protocol, 12(2), e4297. https://doi.org/10.21769/bioprotoc.4297
Menacho-Melgar, Romel, and Michael D. Lynch. “Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.Bio-Protocol 12, no. 2 (January 2022): e4297. https://doi.org/10.21769/bioprotoc.4297.
Menacho-Melgar, Romel, and Michael D. Lynch. “Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.Bio-Protocol, vol. 12, no. 2, Jan. 2022, p. e4297. Epmc, doi:10.21769/bioprotoc.4297.

Published In

Bio-protocol

DOI

EISSN

2331-8325

ISSN

2331-8325

Publication Date

January 2022

Volume

12

Issue

2

Start / End Page

e4297

Related Subject Headings

  • 32 Biomedical and clinical sciences
  • 31 Biological sciences