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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
Author(s) -
MenachoMelgar Romel,
Moreb Eirik A.,
Efromson John P.,
Yang Tian,
Hennigan Jennifer N.,
Wang Ruixin,
Lynch Michael D.
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27444
Subject(s) - autolysis (biology) , lysis , lysozyme , periplasmic space , endonuclease , rna , dna , biology , biochemistry , escherichia coli , microbiology and biotechnology , nucleic acid , enzyme , gene
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.