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  • Protein Expression using the K. lactis Protein Expression Kit - Growth of strains for detection of sereted protein

    Protocol

    1. From the patch of each strain that contains an integrated expression fragment, harvest cells from an area approximately 2 mm2 by scraping with a sterile pipette tip and resuspend the cells in 2 ml of YPGal medium in a sterile culture tube.  Incubate the cultures with shaking (~250 r.p.m.) at 30oC.

      The duration of growth will vary depending on the protein being secreted.  As a general rule, allow for a minimum of 2 days growth at 30oC to obtain a saturated culture (a culture density of >30 OD600 units/ml).  Analysis of culture supernatant may be performed each day thereafter to determine the optimum growth time to achieve maximum secretion of the protein of interest.  Culture sizes will ultimately depend on the desired application.  For example, to determine the efficiency of secretion on previously untested cells, 2 ml cultures will allow for simultaneous analysis of many strains.  Larger cultures (e.g., ≥ 1 L) for protein purification should be inoculated 1:100 with a starter culture grown overnight at 30oC.
    2. Microcentrifuge 1 ml of each culture for 30 seconds to pellet cells.  Remove the culture supernatant to a fresh microcentrifuge tube and store on ice.
    3. Since expression levels of recombinant proteins secreted from K. lactis vary from protein to protein, culture supernatant samples must be analyzed to determine if the protein of interest is being secreted.  Poly-acrylamide gel electrophoresis followed by Coomassie or silver staining of unconcentrated culture supernatant (15 μl per lane) allows for visual detection of proteins that are highly secreted ( e.g. > 10 mg/l).  Alternatively, Western blotting can detect lesser quantities of secreted protein.  If an antibody to your protein of interest is unavailable, an antibody epitope tag (e.g. hemagglutinin (HA) peptide epitope) can be engineered as a C-terminal fusion to the protein of interest.  Finally, if the protein of interest is an enzyme, culture supernatant may be analyzed for the presence of the protein directly by an activity assay.  In such cases it is important to note that the absence of an enzyme activity in culture supernatant does not always indicate a lack of secretion.  For example, the enzyme may be secreted in an inactive form, or the nutrient rich growth medium may inhibit the activity assay.  In such cases, absence of secretion of the protein of interest should also be confirmed by SDS-PAGE or Western analysis.
    4. K. lactis cells can be stored at -70oC, suspended in a final concentration of 20% (v/v) sterile glycerol.  For example, 500 μl of a culture of freshly grown cells can be diluted with 500 μl of a sterile 40% glycerol solution (to give a 20% final glycerol concentration).  Alternatively, a scoop of cells from a freshly grown streak on agar medium can be scraped from the plate using a sterile loop or pipet tip and resuspended directly in 20% steril glycerol.  It is important to make sure that all solutions and tubes that contact the cells are sterilized prior to their use.  To revive frozen K. lactis cells contaning an integrated pKLAC2 construct, streak a small aliquot of frozen cells on YCB agar medium supplemented with 5 mM acetamide.  After this initial growth on YCB agar medium, the cells can be grown without selection in rich medium for protein expression.