scholarly journals 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

2020 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Eirik A. Moreb ◽  
John P. Efromson ◽  
Michael D. Lynch
Keyword(s):  
Protein Expression ◽  
Membrane Integrity ◽  
Heterologous Protein Expression ◽  
List Type ◽  
Two Stage ◽  
Microtiter Plates ◽  
Thaw Cycle ◽  
Protein Expression And Purification ◽  
Phosphate Depletion ◽  
Rna Endonuclease

AbstractWe report improved release of recombinant proteins in E. coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the non specific DNA/RNA endonuclease from S. 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. Post cell harvest, the addition of detergent (0.1% Triton-X100) and a single 30 minutes freezer thaw cycle results in > 90% release of protein (GFP). 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.HighlightsAutoinduction of both cell lysis and nucleotide hydrolysis>90 % lysis and DNA degradationStrains are stable to lysis in the absence of phosphate depletion.

scholarly journals Optimization of phosphate-limited autoinduction broth for two-stage heterologous protein expression in Escherichia coli

BioTechniques ◽  
2021 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Jennifer N Hennigan ◽  
Michael D Lynch
Keyword(s):  
Protein Expression ◽  
Small Scale ◽  
Heterologous Protein Expression ◽  
Scale Production ◽  
Two Stage ◽  
Inducible Promoters ◽  
Media Formulation ◽  
Microtiter Plates ◽  
Shake Flasks ◽  
Consistent Performance

Autoinducible, two-stage protein expression leveraging phosphate-inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date, small-scale production using microtiter plates and shake flasks relies on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, the authors develop a simpler media formulation (AB-2) with just a few autoclavable components. AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate the adoption of phosphate-limited two-stage protein expression protocols.

scholarly journals Optimization of Phosphate Limited Autoinduction Broth for 2-Stage Heterologous Protein Expression in E. coli

2021 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Jennifer N Hennigan ◽  
Michael D Lynch
Keyword(s):  
Protein Expression ◽  
Small Scale ◽  
Heterologous Protein Expression ◽  
Scale Production ◽  
Inducible Promoters ◽  
E Coli ◽  
Media Formulation ◽  
Microtiter Plates ◽  
Shake Flasks ◽  
Consistent Performance

Autoinducible, 2-stage protein expression leveraging phosphate inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date small scale production using microtiter plates and shake flasks rely on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, we develop a simpler media formulation (AB-2) with just a few autoclavable components. We show that AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate adoption of phosphate limited 2-stage protein expression protocols.

scholarly journals Scalable, two‐stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

2020 ◽  
Vol 117 (9) ◽  
pp. 2715-2727 ◽  
Author(s):  
Romel Menacho‐Melgar ◽  
Zhixia Ye ◽  
Eirik A. Moreb ◽  
Tian Yang ◽  
John P. Efromson ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Two Stage ◽  
E Coli ◽  
Phosphate Depletion

scholarly journals Improved, scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

2019 ◽  
Author(s):  
Romel Menacho-Melgar ◽  
Zhixia Ye ◽  
Eirik A. Moreb ◽  
Tian Yang ◽  
John P. Efromson ◽  
...  
Keyword(s):  
Protein Expression ◽  
Stationary Phase ◽  
Recombinant Protein Expression ◽  
Cellular Protein ◽  
List Type ◽  
Heterologous Proteins ◽  
Fed Batch ◽  
E Coli ◽  
Dry Cell Weight ◽  
Phosphate Depletion

AbstractWe report the improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of total cellular protein. Initial use of the method in instrumented fed batch fermentations enables cell densities of ∼30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/−0.7 g/L (∼270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μL (384 well plates), 100 μL (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach ∼ 5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.HighlightsStationary phase protein expression results in high titers.Autoinduction by phosphate depletion enables protein titers from 2-8 g/L.Autoinduction has been validated from 384 well plates to instrumented bioreactors.

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