lyticase - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

"Vacuoles were isolated through hydrolysing yeast cell walls using lyticase, recombinantly expressed in Escherichia coli RSB805 (provided Dr. Randy Schekman, Berkeley), and were prepared from a periplasmic supernatant."

Shen, S. H., Chrétien, P., Bastien, L., & Slilaty, S. N. (1991). Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli. The Journal of Biological Chemistry, 266(2), 1058–1063.

rel:///home/bjorn/files/wiki-public-protocols/Protocol_041007_lyticase_prep_from_ecoli.doc

Purification of lyticase

Prep work:

  1. Make assay cells: grow desired WT yeast cells to <1 OD640 (cells do not spheroplast well at higher OD!) harvest cells and wash with dH20 resuspend cells in 50 mM Tris pH 7.4 to OD640 = 5.

  2. Prepare RSB805 (pUV5::DH 5) stationary: LBamp, 37 C

  3. Make COLD "shock" buffer

Induction:

  1. Inoculate LBamp with overnight stationary; 1/100 dilution is good... even 1/200 is OK, but remember that doubling time for this strain is 40 minutes
  2. Let the bugs grow until 0.5 OD640; INDUCE by adding IPTG to 0.4mM
  3. Grow for 5 hours

Shocking: (note: first four steps are done at room temp.)

  1. Harvest cells and wash with 25 mM Tris pH 7.4
  2. Resuspend cells with 1/50 original volume in 25 mM Tris pH 7.4; add EDTA to 2 mM final concentration
  3. Add equal volume of 40% sucrose, 25 mM Tris pH 7.4; mix gently for 20 minutes and no longer!
  4. Spin cells in GSA at 7,500rpm for 10 minutes. for good "shocking", make sure that all sucrose buffer is removed
  5. Resuspend cells with no more than 1/50 original volume in ICE COLD 0.5mM MgSO4; mix gently: 20 minutes on ice
  6. Spin cells in SS34 at 10,000 rpm for 10 minutes
  7. Transfer the supe containing the lyticase to dialysis tubes and dialyze against 50mM NaOAc pH 5.0

Purification:

  1. Batch bind dialyzed supe to CM Sepharose which has been pre-equilibrated in 50mM NaOAc pH5.0. Load bound mix onto fat short column (save the flowthrough just in case...)
  2. Wash the column with 50mM NaOAc pH5.O (2-3x column volume)
  3. Elute lyticase with 150mM NaCl, 50mM NaOAc pH5.0. Flow rate should be ~7ml/minute and collect 7ml fractions. General rule of thumb: lyticase will start to elute just after one column volume of elution buffer has gone through
  4. Monitor every fifth fraction for activity then assay every other fraction across the peak. combine the peak fractions.

Assay:

  1. Make fresh 2x buffer: 100 mM Tris-HCl pH 7.4, 80 mM 2-ME

  2. In each assay point: 1ml 2x assay buffer 920 uL H2O 80 uL cells (final OD640 is about 0.2) 0 - 50 ul collected fractions

  3. Remove 1 ml and measure OD640 for "zero" point

  4. Incubate remainder at 30C for 30 minutes then determine OD640; 1 unit = 10% decrease in OD640 after 30 minutes at 30C

Please note: We normally use crude shock fluid unless purified lyticase is especially desired. To store crude shock fluid (i.e. the supe from step 6, without dialysis or pH adjustment), aliquot and quick freeze in liquid nitrogen and store at -80C. We have not noticed a significant decrease in activity after prolonged storage. It's probably a good idea to add azide to the shock fluid after thawing to keep bugs from growing in it.