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  • pKLAC1-malE Control Plasmid

    Discontinued Date

    01/01/2013
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    Categories:
    Discontinued Products

    Description

    pKLAC1-malE is a control plasmid for the K. lactis Expression Kit (NEB #E1000). The backbone integrative expression vector, pKLAC1, directs high-level expression of recombinant protein from the yeast Kluyveromyces lactis. Vector pKLAC1-malE carries the E. coli malE gene, encoding the maltose binding protein (MBP). pKLAC1-malE can be used to test plasmid transformation, cell culture, and protein production procedures. A typical K. lactis GG799 strain containing integrated pKLAC1-malE will produce > 25 mg/L of MBP (Figure 1).

    Figure 1: SDS-polyacrylamide gel electrophoresis of secreted recombinant maltose binding protein (MBP). (A) Detection of secreted MBP by Coomassie staining. Lane 1: Protein Molecular Weight Markers. Lane 2: spent culture medium (15 µl) from wild-type K. lactis cells. Lane 3: spent culture medium (15 µl) from K. lactis cells harboring integrated pKLAC1-malE.(B) Detection of secreted MBP by Western analysis using a polyclonal anti-MBP antibody (NEB #E8031S; 1:2000 dilution) and an anti-rabbit IgG-HRP secondary antibody (1:2000 dilution). Lane 1: culture medium from K. lactis cells (15 µl). Lane 2: culture medium (15 µl) from K. lactis cells containing integrated pKLAC1-malE.

    Highlights

    • Expression of the fusion gene is under the control of the strong K. lactis PLAC4-PBI promoter
    • K. lactis strain GG799 can be used as an expression host
    • Ampicillin resistance for propagation in E. coli (See notes for strain recommendations.)
    • Fungal acetamidase selectable marker for integration into the K. lactis genome

    Properties and Usage

    Storage Temperature

    -20°C

    Notes

    1. NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987), NEB 5-alpha Electrocompetent E. coli (NEB #C2989) and NEB 5-alpha Competent E. coli (Subcloning Efficiency) (NEB #C2988) are all recommended for propagation of this control plasmid.

    References

    1. Colussi, P.A. and Taron, C.H. Unpublished observation

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Interactive Tools

    Application Notes

    Always do the control transformation. This will help mediate any downstream expression issues which may arise.