Product Description Simplify your protein expression and purification today! Roke’s Autoinduction/Autolysis E. coli strain is a proprietary bacterial strain that has been engineered for routine protein expression. Protein autoinduction simplifies expression and results in high protein titers while also providing cell autolysis and nucleotide autohydrolysis capabilities to facilitate routine downstream workflows. Our strain leverages two-stage protein expression where cells grow until phosphate in the media becomes depleted. Once low phosphate conditions are reached, expression of the protein of interest is driven by the activation of low phosphate inducible promoters. This results in decoupling cell growth from protein expression, enabling the use of standard bioprocesses for different proteins of interests and therefore removing the need to monitor cell growth or optimize induction conditions. In addition to the protein of interest, two chromosomally integrated accessory proteins are co-expressed: a lysozyme expressed in the cytosol (λR gene from bacteriophage lambda) and a nuclease expressed in the periplasm (nucA gene from Serratia marcescens). After cell harvesting, addition of a low amount of detergent (0.1% v/v) coupled with a freeze-thaw cycle results in >90% protein release and complete host oligonucleotides removal. Technical Information Host 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) Plasmid (Control only) pHC-Kan-yibDp-mcherry (here): high copy plasmid for the low phosphate auto-induction of mcherry, confers kanamycin resistance. For the plasmid map, click here Antibiotic Resistance Apramycin, Gentamicin (and Kanamycin for strain bearing control plasmid) Product Manual Click here Key References 1. Menacho-Melgar, R., Moreb, EA., Efromson, JP., Yang, T., Hennigan, JN., Wang, R., and Lynch, MD., 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. Biotechnology & Bioengineering., 28 May 2020. 2. Menacho-Melgar, R., Ye, Z., Moreb, EA., Yang, T., Efromson, JP., Decker, JS., Wang, R., and Lynch, MD., Scalable, two‐stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion. Biotechnology & Bioengineering., 22 May 2020. 3. Menacho-Melgar, R., Hennigan, JN., and Lynch MD. Optimization of phosphate-limited autoinduction broth for two-stage heterologous protein expression in Escherichia coli. Biotechniques, Available Online August 25, 2021.
