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Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease.

Publication ,  Journal Article
Menacho-Melgar, R; Moreb, EA; Efromson, JP; Yang, T; Hennigan, JN; Wang, R; Lynch, MD
Published in: Biotechnology and bioengineering
September 2020

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.

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Published In

Biotechnology and bioengineering

DOI

EISSN

1097-0290

ISSN

0006-3592

Publication Date

September 2020

Volume

117

Issue

9

Start / End Page

2852 / 2860

Related Subject Headings

  • Recombinant Proteins
  • RNA
  • Protein Engineering
  • Muramidase
  • Hydrolysis
  • Escherichia coli
  • Deoxyribonuclease I
  • DNA
  • Biotechnology
  • Bioreactors
 

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Menacho-Melgar, R., Moreb, E. A., Efromson, J. P., Yang, T., Hennigan, J. N., Wang, R., & Lynch, M. D. (2020). Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease. Biotechnology and Bioengineering, 117(9), 2852–2860. https://doi.org/10.1002/bit.27444
Menacho-Melgar, Romel, Eirik A. Moreb, John P. Efromson, Tian Yang, Jennifer N. Hennigan, Ruixin Wang, and Michael D. Lynch. “Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease.Biotechnology and Bioengineering 117, no. 9 (September 2020): 2852–60. https://doi.org/10.1002/bit.27444.
Menacho-Melgar R, Moreb EA, Efromson JP, Yang T, Hennigan JN, Wang R, et al. Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease. Biotechnology and bioengineering. 2020 Sep;117(9):2852–60.
Menacho-Melgar, Romel, et al. “Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease.Biotechnology and Bioengineering, vol. 117, no. 9, Sept. 2020, pp. 2852–60. Epmc, doi:10.1002/bit.27444.
Menacho-Melgar R, Moreb EA, Efromson JP, Yang T, Hennigan JN, Wang R, Lynch MD. Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease. Biotechnology and bioengineering. 2020 Sep;117(9):2852–2860.
Journal cover image

Published In

Biotechnology and bioengineering

DOI

EISSN

1097-0290

ISSN

0006-3592

Publication Date

September 2020

Volume

117

Issue

9

Start / End Page

2852 / 2860

Related Subject Headings

  • Recombinant Proteins
  • RNA
  • Protein Engineering
  • Muramidase
  • Hydrolysis
  • Escherichia coli
  • Deoxyribonuclease I
  • DNA
  • Biotechnology
  • Bioreactors