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 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.

Instant Taq: Rapid Autoinducible Expression and Chromatography-free Purification of Taq polymerase

“…9,15 This plasmid was transformed into strain DLF_R004 (Genotype: F-, λ-, Δ (araD-araB)567, lacZ4787(del)(::rrnB-3), rph-1, Δ(rhaD-rhaB)568, hsdR51, ΔackA-pta, ΔpoxB, ΔpflB, ΔldhA, ΔadhE , ΔiclR, ΔarcA, ΔompT::yibDp-ƛR-nucA-apmR ), which has been engineered for the low phosphate induction of a periplasmic nuclease ( S. marcescens nucA, benzonase™ ) and cytoplasmic Lambda phage lysozyme as illustrated in Figure 1a. 11 After the disruption of the membrane in the presence of 0.1% triton-X, lysozyme degrades the peptidoglycan cell wall and the nuclease degrades residual cellular DNA and RNA (Figure 1b). 11 Cell growth and autoinduction of Taq expression is performed in autoinduction broth as described by Menacho-Melgar et al 10,14 Using this system, Taq was expressed to ~35% of the total cellular protein approaching titers of 900 mg/L as seen in Figure 2a.…”

“…11 After the disruption of the membrane in the presence of 0.1% triton-X, lysozyme degrades the peptidoglycan cell wall and the nuclease degrades residual cellular DNA and RNA (Figure 1b). 11 Cell growth and autoinduction of Taq expression is performed in autoinduction broth as described by Menacho-Melgar et al 10,14 Using this system, Taq was expressed to ~35% of the total cellular protein approaching titers of 900 mg/L as seen in Figure 2a.…”

Dynamic control over feedback regulation identifies pyruvate-ferredoxin oxidoreductase as a central metabolic enzyme in stationary phaseE. coli

“…3,4,[15][16][17][18] Induction is implemented using phosphate depletion as an environmental trigger. [19][20][21][22] The native E. coli Type I-E Cascade/CRISPR system is used for gene silencing (Figure 2b). 16,23 Targeted proteolysis is implemented by linking the expression of the chaperone SspB to phosphate deprivation.…”

Low-Cost, Large-Scale Production of the Anti-viral Lectin Griffithsin

“…On the other hand, this simple analysis cannot capture all the complexities and interdependencies of a pharmaceutical process. The two most promising possibilities may be increases in GRFT expression or the use of engineered host strains that reduce DNA in lysates ( Menacho-Melgar et al, 2020a ). Either of these changes may reduce the number of polishing operations required after precipitation.…”