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  • K. lactis Protein Expression Kit

    Description

    The K. lactis Expression Kit provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis (Figure 1). Proteins may be produced intracellularly or be secreted using the supplied integrative expression vector pKLAC2 (Figure 3). To achieve protein secretion, a gene of interest is cloned downstream of the K. lactis α-mating factor secretion domain (α-MF; Figure 4) which is eventually processed in the Golgi resulting in secretion of the desired protein (Figure 1).

    The K. lactis expression system offers several advantages over other yeast and bacterial protein expression systems. First, K. lactis has been used to produce proteins at industrial scale in the food industry for over a decade due to its ability to rapidly achieve high culture densities and abundantly produce recombinant proteins (Figure 2). Second, yeast expression is driven by a variant of the strong LAC4 promoter that has been modified to lack background expression in E. coli (1). Therefore, genes toxic to E. coli can be cloned into pKLAC2 in bacteria prior to their expression in yeast. Third, the kit includes highly competent K. lactis cells making the technology easy-to-use for those not accustomed to working with yeast. Their high transformation efficiency makes the system suitable for methods that require large numbers of transformants, for example, expression cloning using cDNA libraries. Selection of yeast transformants uses a unique antibiotic-free method in which acetamidase (amdS) expressed from pKLAC2 permits transformed cells to utilize acetamide as a sole nitrogen source on defined medium. Acetamide selection promotes formation of cells containing multiple integrations of pKLAC2 which results in higher yields of protein. Finally, proteins expressed in K. lactis have access to eukaryotic protein folding and glycosylation machinery that E. coli cells do not possess, making it an important alternative to bacterial expression systems.

    Figure 1:
    Secreted protein processing. In the nucleus, an integrated expression vector encoding a fusion between the α-MF domain (blue) and a desired protein (black) is expressed. A signal peptide in the α-MF domain directs entry of the fusion protein into the endoplasmic reticulum (ER) and is removed by signal peptidase (SP). The fusion protein is transported to the Golgi where the Kex protease removes the α-MF domain. The protein of interest is then secreted from the cell.
    Figure 2:
    SDS-polyacrylamide gel electrophoresis separation of secreted recombinant maltose binding protein (MBP) and detection 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 an integrated expression cassette containing the E. coli malE gene.

    Figure 3: The pKLAC2 expression vector. pKLAC2 (9107 bp) contains the 5´ and 3´ ends of the LAC4 promoter (PLAC4-PBI) separated by DNA encoding β-lactamase (ApR) and the pMB1 origin (ori) to allow for its propagation in E. coli. The K. lactis α-mating factor secretion leader sequence (α-MF), multiple cloning site (MCS), and the LAC4 transcription terminator (TT) lie immediately downstream of 3´ PLAC4-PBI. The yeast ADH1 promoter (PADH1) drives expression of an acetamidase selectable marker gene (amdS). The vector can be linearized by digestion with SacII or BstXI to create a linear DNA fragment capable of inserting into the native LAC4 promoter region of the K. lactis genome.


    Figure 4:
    pKLAC2 multiple cloning site. pKLAC2 (9107 bp) contains the K. lactis a-mating factor secretion leader sequence (blue background) and a polylinker immediately downstream of the PLAC4-PBI promoter. Unique polylinker restriction sites are indicated. Half-arrows show the positions of pKLAC-series vector-specific sequencing primers available from New England Biolabs.

    DNASU is a central repository for plasmid clones and collections that may also be helpful.

    Highlights

    • Supplied with competent K. lactis cells
    • Easy-to-use protocols for those inexperienced with yeast systems
    • Attractive commercial sublicensing
    • Clone and express genes toxic to E. coli
    • No expensive antibiotics or methanol required

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pKLAC1-malE Control Plasmid-201,000 μg/ml
    pKLAC2 Vector-201,000 μg/ml
    SacII-2020,000 units/ml
    Integration Primer 110X
    Integration Primer 210X
    Integration Primer 310X
    NEB Yeast Transformation Reagent (5 ml)1X
    Acetamide solution (sterile) (10 ml)100X
    Yeast Carbon Base Medium (12g)
    NEBuffer 4-2010X
    K. lactis GG799 Competent Cells-805 reactions

    Advantages and Features

    Features

    • High yield protein expression
    • Rapid high cell density growth
    • Supplied with competent K. lactis cells
    • Easy-to-use protocols for those inexperienced with yeast systems
    • Attractive commercial sublicensing

    Properties and Usage

    Storage Temperature

    -80°C

    Shipping Notes

    • Ships on dry ice

    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 and subcloning pKLAC1 control plasmid and pKLAC2 vector.

    References

    1. Colussi, P.A. and Taron, C.H. (2005). Appl. Environ. Microbiol.. 71
    2. van Ooyen, A.J.J.et al. (2006). FEMS Yeast Research . 6, 381-392.
    3. Read, J.D.et al. (2007). Appl. Environ. Microbiol.. 73, 5088-5096.
    4. Ekborg, N.A.et al. (2007). Appl. Environ. Microbiol.. 73, 7785-7788.
    5. Platko, J.D.et al. (2008). Protein Expr. Purif.. 57, 57-62.

    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.

    Manuals

    The Product Manual 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 these document files: manual[Catalog Number].
    1. What systems does NEB offer for protein expression and purification?
    1. Protein expression using the K. lactis Protein Expression Kit - Linearization of pKLAC2 for integrative transformation of K. lactis.
    2. Protein Expression using the K. lactis Protein Expression Kit - Identification of Multi-copy Integrants
    3. Protein Expression using the K. lactis Protein Expression Kit - Identification of properly integrated cells
    4. Protein Expression using the K. lactis Protein Expression Kit - Simultaneous Expression of Multiple Proteins
    5. Protein expression using the K. lactis Protein Expression Kit - Cloning a PCR fragment into pKLAC2.
    6. Protein Expression using the K. lactis Protein Expression Kit - Growth of strains for detection of sereted protein
    7. Protein Expression using the K. lactis Protein Expression Kit - Transformation of K. lactis GG799 cells

    Selection Tools

    Application Notes

    K. Lactis system (E1000S)

    Gibson assembly (E2611) is an excellent way to insert a PCR fragment containing your gene into a pMAL vector. For pMAL-c5X, add the sequence (5'dGGGATCGAGGGAAGG