Home Protocols Protein Expression using the K. lactis Protein Expression Kit - Identification of Multi-copy Integrants

Protein Expression using the K. lactis Protein Expression Kit - Identification of Multi-copy Integrants

Overview

It is possible for up to 10 copies of the expression casette to tandemly insert into the genome during transformation.  Strains harboring multiple integrations often produce more secreted protein.  An advantage of selection for K. lactis transformants on YCB Agar Medium containing acetamide is that it enriches for cells harboring multiple tandem integrations.  Multiply integrated cells can be identified using whole-cell PCR with Integration Primers 2 and 3 to amplify a 2.58 kb product.

Protocol

  1. For each transformant patched and grown on YCB Agar Medium plates containing 5 mM acetamide, harvest cells from an area approximately 1 mm2 by scraping with a pipette tip and resuspending the cells in 100 μl of 0.2 M LiAc containing1 % SDS.
  2. Heat at 70°C for 15 minutes then add 300 μl 100% ethanol and vortex to mix.
  3. Centrifuge the cells at 15,000 x g for 3 minutes at room temperature.
  4. Carefully remove the supernatant and air dry the pellet for 10 minutes.
  5. Resuspend the pellet in 50 μl TE (10 mM Tris-HCl, pH 7.5 containing 1 mM EDTA).
  6. Centrifuge to remove any insoluble material for 30 seconds at 13,000 x g.
  7. Transfer the supernatant to a fresh tube and keep on ice. Use 1 μl as a template for a 50 μl PCR reaction as follows: 

    3.2 μl 10X Integration Primer 2 (7.8 μM stock)
    3.2 μl 10X Integration Primer 3 (7.8 μM stock)
    25 μl Q5 High-Fidelity 2X Master Mix (NEB #M0492)
    1.0 μl LiAc/SDS treated supernatant from Step 7 above
    17.6 μl - deionized water
    ----------------------------------------------------------------------------------
    50 μl - final reaction volume

  8. Thermocycling should consist of an initial denaturation at 98°C for 30 seconds followed by 30 rounds (98°C for 10 seconds, 60°C for 30 seconds and 72°C for 75 seconds) and a final extension at 72°C for 2min. 
  9. Analyze 10 μl of each amplification reaction on a 1% agarose gel.
    Cells harboring multiple tandem integrations of the expression fragment at the LAC4 locus in the K. lactis genome will result in amplification of a 2.58 kb product.
  10. Test strains harboring multiple copies of the expression fragment for secretions of the protein of interest.