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Product Description
Roke’s 6AL E. coli strain is a proprietary bacterial strain that has been engineered to express high titers of nanobodies 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. Upon entry into stationary phase, targeted depletion of TrxB changes the redox cytoplasmic potential facilitating the formation of disulfide bonds. 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.
A snapshot of strain performance is shown below, highlighting nanobody titers from 6AL expression in microfermentations at 37°C. Notably, the dashed lines represent the highest reported periplasmic and cytoplasmic expression levels of VHH in E. coli before 6AL.
Adapted from: Hennigan, JN., Menacho-Melgar, R., Sarkar, P., Golovsky M., and Lynch MD. Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control. Metab Eng. 24 Jul 2024.
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, , trxB-das4-tetR, Δgor-phoA-dsbC_purR) |
| Plasmids |
(Controls only) |
| Antibiotic Resistance | Apramycin (and Kanamycin for strains 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. Hennigan, JN., Menacho-Melgar, R., Sarkar, P., Golovsky M., and Lynch MD. Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control. Metab Eng. 24 Jul 2024. |