XS™ E. coli Expression Systems

  • Introduction

    Due to the historical success of E. coli for the expression of recombinant proteins, this host type forms the foundation of Lonza's XS™ Technology Toolbox. Our E. coli Systems are based on the commonly accepted K-12 and B microbial strains. The XS™ E. coli offering includes 3 different means of induction:  sugar, IPTG and nutrient depletion, leveraging different regulatory mechanisms:  positive and negative exponential, and positive stationary phase.  This diverse set of options increases the probability of expression success for a variety of recombinant proteins.

     

    Lonza's XS™ E. coli Systems Include:

    • A Shared Plasmid Backbone which enables the efficient use of our high throughput cloning platform for rapid strain development
    • Complementary E. coli Systems to increase the probability of expression success
    • 100% Plasmid Stability  which improves process robustness and delivers higher titers
    • Platform fed-batch fermentation and recovery protocols which are tailored to each XS™ promoter system to form the foundation of a robust and scalable upstream process

     

    Plasmid Stability

    Plasmid stability is a common concern when expressing complex proteins in microbial systems. Lonza’s auxotrophic selection system achieves 100% plasmid stability throughout the fermentation process via the deletion of an essential gene in our dedicated E. coli strains. This gene encodes a protein essential to cell survival.  This gene is complemented in Lonza’s upgraded XS™ plasmid backbone to ensure that only cells that have retained the plasmid will survive and produce target protein.


    An example of a typical E. coli primary screen of a difficult-to-express protein is shown in the figure below.

     

     Figure 1.  The productivity distribution of over 230 individual E. coli clones with expression ranging from 5 to 160 mg/mL.

  • Sugar Inducible

    One of our most popular XS™ Technologies is Lonza’s E. coli Sugar Inducible System. This System is induced by either rhamnose or melibiose and includes tightly-regulated expression where high cell density is achieved without premature induction.  This results in higher titers (see Figure 2). In addition, the system features tunable expression, where very slow induction kinetics favors the production of soluble, functional target protein, especially for periplasmic production (see Figure 3). Lonza's patented sugar-inducible systems have successfully been used in over 30 customer projects and are capable of achieving expression levels in excess of 20 g/L, a 10-20 fold improvement compared to a commercially available E. coli system.  This proven technology has been shown to successfully express many types of products, most notably, difficult-to-express proteins.

    Lonza's XS™ E.coli Sugar Inducible System is available under a Research Evaluation Agreement.

     

    Sugar Inducible System Components Include:

    • 5  E. coli strains
    • Generic plasmid backbone with stabilizing elements
    • 2 Promoters
    • Numerous secretion signal options
    • Options for monocistronic or polycistronic expression
    • Same multi-cloning site for all plasmids
    • Options for intracellular or periplasmic expression

     

     

    Figure 2. Fed-batch fermentation of a sugar-induced XS™ E. coli strain producing an intracellular target protein reaching a maximum titer in excess of 20 g/L. (DCW, dry cell weight)

     

     

    Figure 3.  The customer and Lonza's production strains were cultivated in side-by-side 10 L fed-batch fermentation runs. The customer’s strain produced little insoluble product which required a poorly-yielding and laborious refolding process. The Lonza XS™ strain delivered 15 times more product than the customer’s strain, due to significantly higher soluble expression while eliminating the need for a refolding step.

  • Depletion Inducible

    Lonza's E. coli Depletion Inducible System controls induction by limiting a key metabolite. This system contains an array of different strength promoters which enables the fine tuning of the expression rate, maximizing soluble expression (see Figure 4 below).  This is in contrast to Lonza’s Sugar Inducible System, where expression tuning is performed using different sugar/inducer concentrations. In addition, the Depletion Inducible System expresses protein during the stationary phase of cell growth which expands the design space for experimentation in E. coli.

     

    Depletion Inducible System Components Include:

    • E. coli strains
    • Generic plasmid backbone with stabilizing elements
    • Numerous promoters
    • Numerous secretion signal options 
    • Options for monocistronic or polycistronic expression
    • Same multi-cloning site for all plasmids (Shared plasmid backbone)
    • Options for intracellular or periplasmic expression

     

     

       Empty Plasmid               pPKL105.1                       pOCL5                            pOCL3                         pBGL23

      Empty Promoter                Variant 1                        Variant 2                          Variant 3                       Variant 4
     Promoter Strength       Promoter Strength       Promoter Strength       Promoter Strength      Promoter Strength
                0.5                                     7                                   100                                  218                               238

    Figure 4.  The amount of GFP is visualized by green fluorescence (after UV light exposure) and shows the strength of each promoter.

     

    High Cell Density

    A strong feature of Lonza's Depletion Inducible System is tightly-regulated expression. High cell density is achieved without premature induction, resulting in higher titers (see Figure 5 below).

     

    Figure 5.  A high cell density fermentation run using the depletion inducible expression system is shown where the productivity is peaking after about 70 hours with 11.6 g/L of the target protein.

  • IPTG Inducible

    Lonza's  E. coli Toolbox is complemented by a set of well-accepted and well-known expression options, all based on the use of IPTG as inducer.   We offer the T5 phage promoter which drives very high titers with very short induction times and optionally the lac and tac promoters, which are still used in commercial production processes.  When coupled with our proprietary XS™ strains and vectors this system generates highly stable commercially-viable cGMP strains.

    The IPTG Inducible System complements our other XS™ E. coli Systems and is typically tested as part of an overall expression feasibility study.  Screening all three XS™ E.coli systems in parallel increases the probability of establishing the best production clone for your product. 

     

    IPTG Inducible System Components Include:

    • 2 E. coli strains
    • Generic plasmid backbone with stabilizing elements
    • Numerous secretion signal options  
    • Options for monocistronic or polycistronic expression
    • Same multi-cloning site for all plasmids (shared plasmid backbone)
    • Options for intracellular or periplasmic expression

     

    Figure 6.  The productivity and Dry Cell Weight (DCW) over time for an IPTG-induced XS™ strain producing an intracellular target protein.