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.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Menacho-Melgar, R; Moreb, EA; Efromson, JP; Yang, T; Hennigan, JN; Wang, R; Lynch, MD

Published Date

  • May 28, 2020

Published In

PubMed ID

  • 32462658

Pubmed Central ID

  • 32462658

Electronic International Standard Serial Number (EISSN)

  • 1097-0290

International Standard Serial Number (ISSN)

  • 0006-3592

Digital Object Identifier (DOI)

  • 10.1002/bit.27444

Language

  • eng