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

Abstract

We 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. Highlights :• Autoinduction of both cell lysis and nucleotide hydrolysis • >90 % lysis and DNA degradation • Strains are stable to lysis in the absence of phosphate depletion.

Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in E. coli

“…We have reported two-stage autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion. , This methodology utilizes engineered host strains, Autoinduction Broth (AB), and a truncated, low phosphate inducible (PhoB activated) promoter from the yibD gene . This promoter (Figure f) had similar expression levels in rich AB as well as minimal media and scaled successfully in instrumented bioreactors .…”

Robustness testing and scalability of phosphate regulated promoters useful for two-stage autoinduction inE. coli

“…For example, the ytfK promoter had a 60% reduction in expression in minimal media as compared to AB, while the tktB promoter had an almost 400% improvement in expression in minimal media. We next compared the behavior of the modified (truncated) yibD promoter 10,11 with the full length native promoter ( Figure 2(e-j)). While both promoters had similar behavior in AB media, the full length promoter had a significant decrease (over 50% reduction) in expression in minimal media, presumably due to the presence of the extra NsrR repressor binding site or additional uncharacterized regulation.…”

“…We have reported two-stage autoinduction of recombinant proteins in E. coli utilizing phosphate depletion, which can simplify expression protocols and lead to elevated protein titers. 10,11 This methodology utilizes engineered E. coli host strains as well as optimized Autoinduction Broth (AB) and a low phosphate inducible promoter derived from the E. coli yibD ( waaH ) gene promoter. 12,13 This promoter led to similar expression levels in both rich AB as well as minimal mineral salts media in instrumented bioreactors.…”