XS™ Pichia Expression Systems

  • Introduction

    Although E. coli host systems remain the industry workhorse, there are times when an ideal expression outcome for your product is not achieved.  In these situations, we offer an alternative approach to microbial expression with a range of Pichia pastoris host systems.

    As a eukaryotic host organism, Pichia is able to effectively express small polypeptides, as well as large, complex proteins that require higher order post translational modifications. In addition, Pichia uses a signal sequence, which helps it to express soluble secreted product as opposed to undesirable intracellular inclusion bodies. Soluble secreted product, combined with use of minimal media makes recovery and downstream processing easier, more efficient, and improves overall process yields. Pichia also lacks endotoxin, therefore eliminating this typical purification challenge.

    Lonza has established a proprietary Pichia host which has been developed for excellent growth performance (see Figure 1 below). Our XS™ Pichia toolbox includes four different promoter systems: Methanol Inducible, Glucose Regulated, Strong Constitutive, and GAP Constitutive. This range of promoter options provides complementary mechanisms for the optimal expression of your product.  In addition, the toolbox also includes: novel signal peptides, zeocin selection marker, two chromosomal integration sites, and single or multi-copy insertion options.

    Lonza's XS™ Pichia Systems Include:

    • Strong promoters that deliver high titers 
    • Novel signal peptide that eliminates N-terminal variant formation
    • Soluble secreted expression simplifying downstream processing
    • Tunable copy number that increases the probability of creating the best production clone
    • Complementary Pichia systems increase the probability of expression success

    Figure 1.  Wet cell weight (WCW) and dry cell weight (DCW) were measured over time.  Similar growth and fermentation profiles were seen for both the Lonza host strain and an industry standard. The measured WCW and DCW concentrations were reproducible.

     

     

  • Methanol Inducible

    Pichia is a methylotrophic yeast, capable of metabolizing methanol as its sole carbon source via the AOX1 promoter. This expression mechanism has been the most widely-used Pichia system in the industry. For reasons described on the Glucose Regulated page (see tab on right), methanol is not an ideal inducer.  In some cases however, the advantages of higher titers may outweigh the issues related to methanol.

    Lonza has combined its proprietary XS™ Pichia host and signal sequences with this proven promoter system to offer customers a comprehensive Pichia expression toolbox. Multi-gram-per-liter titers have been achieved with this system (see Figure 2 below).

    This system is available for use in your own labs under our Research Evaluation Agreement.

     

    Methanol Inducible System Includes:

    • Host: Proprietary Pichia pastoris LP1
    • Proprietary Plasmid backbone: pXSP
    • Proprietary signal peptides
    • Same multi-cloning site for all plasmids
    • Selection Marker: zeocin
    • E. coli type origin of replication
    • Single or multi-copy insertion possible at 2 different chromosomal loci
    • Fed-batch fermentation platform protocols

     

    Figure 2. 10L fed-batch fermentation of a protein using the XS™ Pichia Methanol Inducible System showing titers in excess of 9 g/L. 

  • Glucose Regulated

    Inducible promoters are beneficial for the production of heterologous proteins that interfere with cell metabolism and the viability of the cells. Using inducible promoters, the growth phase can be decoupled from the production phase. This enables high cell densities to be achieved prior to product expression leading to high titers (see Figure 3 below).

    The AOX1 promoter is widely used in the industry and is induced by methanol requiring volatile solvent handling. Methanol feeds need to be carefully controlled since methanol accumulation during fermentation is toxic to the Pichia cells. In addition, methanol induces a stress response which can ultimately lead to target protein degradation. Lonza’s proprietary inducible Pichia system avoids methanol and uses glucose as a trigger for induction.

    This system is available for use in your own labs under our Research Evaluation Agreement.

     

    Glucose Regulated System Includes:

    • Host: Proprietary Pichia pastoris LP1
    • Proprietary Plasmid backbone: pXSP
    • Proprietary signal peptides
    • Same multi-cloning site for all plasmids
    • Selection Marker: zeocin
    • E. coli type origin of replication
    • Single or multi-copy insertion possible at 2 different chromosomal loci
    • Fed-batch fermentation platform protocols

     

    Figure 3.  10L fed-batch fermentation of a target protein using the XS™ Pichia Glucose Regulated Promoter System.  Expression levels in excess of 1 g/L were achieved.

  • Strong Constitutive

    For products that do not interfere with Pichia metabolism and cell viability, non-induced constitutive can be an attractive expression strategy. Lonza has developed a proprietary promoter which consistently achieves higher titers than the commonly used GAP Constitutive promoter (see Figure 4 below).  An additional benefit of this system is that it does not require the use of methanol.

     

    Strong Constitutive System Includes:

    • Host: Proprietary Pichia pastoris LP1
    • Proprietary Plasmid backbone: pXSP
    • Proprietary signal peptides
    • Same multi-cloning site for plasmids
    • Selection Marker: zeocin
    • E. coli type origin of replication
    • Single or multi-copy insertion possible at 2 different chromosomal loci
    • Fed-batch fermentation platform protocols

     

    Figure 4. 10L fed-batch fermentation of a therapeutic protein using XS™ Pichia Strong Constitutive System versus a commercially available GAP Constitutive promoter system using strains with the same gene copy.  Twice the expression of the target protein was achieved with the Lonza Strong Constitutive System.

  • GAP Constitutive

    Vectors containing a constitutive Pichia promoter derived from the glyceraldehyde-3-phosphate dehydrogenase gene (GAP), are widely known and used in the industry. For products that do not interfere with Pichia metabolism and cell viability, non-induced constitutive can be an attractive expression strategy.

    Lonza has combined this promoter with our novel XS™ Pichia host and signal sequences to offer customers another complementary Pichia option to screen.  This system has shown productivity levels in excess of 1 g/L for a novel therapeutic candidate (see Figure 5 below).  An additional benefit of this system is that it does not require the use of methanol.

    This system is available for use in your own labs under our Research Evaluation Agreement.

     

    GAP Constitutive System Includes:

    • Host: Proprietary Pichia pastoris LP1
    • Proprietary Plasmid backbone: pXSP
    • Proprietary signal peptides
    • Same multi-cloning site for all plasmids
    • Selection Marker: zeocin
    • E. coli type origin of replication
    • Single or multi-copy insertion possible at 2 different chromosomal loci
    • Fed-batch fermentation platform protocols

     

    Figure 5. 10L fed-batch fermentation of a novel therapeutic candidate using XS™ Pichia GAP Constitutive System showing titers in excess of 1 g/L.