Expression and solubility of overexpressed proteins


Please see the Qiagen booklet for a detailed description of the procedure.

Transformation and expression

  • Transform E.coli BL21DE with the expression plasmid. Plate the transformation mix on a selective plate (as back-up) and inocculate 100ml LB. Grow overnight at 37°C. The culture will usually reach an OD600=3-5
  • inocculate 50 ml LB+Amp (pre-warmed) with 2.5 ml of the fresh overnight culture to an OD=0.3 and shake for 1 h until the density is about 0.5-0.8.
  • take a 1 ml sample, measure the OD280 and spin for 1 min at 13k. Suspend the cells in LŠmmli to 1OD per 100 ul. (use 50 ul if the OD600=0.5). Load 5 ul of this sample on SDS-PAGE (0 h point).
  • induce protein expression by adding 50 ul IPTG to 1 mM (stock is 1 M)
  • grow for 4h, 37C
  • At 1 h, 2h, 3h after induction take a 1 ml sample, measure OD600, spin, suspend the cells in Lämmli buffer (50 µl for OD=1 )

Lysis

  • After 4 h collect cells in a 50 ml falcon tube  (wet weight ca. 1 g). Spin for 10 min at 5k.
  • suspend in 5 ml lysis buffer in a 15 ml Falcon tube. Add lysozyme to 1 mg/ml and incubate on ice for 30 min. Alternatively, freeze in liquid nitrogen and thaw.
  • sonicate 6 x 10s, keep on ice water (for better heat exchange), suspension should clarify (max. power, 100% pulse), the tube may slightly warm up.
  • spin for 20 min at 5k, 4°C, supernatant is soluable fraction
  • suspend the pellet in 5 ml lysis buffer,
  • repeat sonication, spin 20 min, 4°C, discard second supernatant
  • pellet is the insoluable fraction
  • suspend pellet in about 1 ml Lämmli (volume depends on initial OD600)

binding to beads and elution

  • work in cold room/on ice with the soluable fraction
  • suspend 20 µl of Ni beads or GSH beads in 500 µl lysis buffer, briefly spin (2k, 1min), pipette the suspension of beads with a decapitated tip
  • carefully remove the supernatant, add 100 ul of lysis buffer and transfer to the tube with the soluable fraction
  • incubate on a wheel for 15-30 min at 4°C
  • briefly spin, supernatant is unbound fraction, take 20 µl + Lämmli
  • suspend beads in 500 µl of lysis or wash buffer
  • briefly spin (2k, 1 min, 4°C)
  • repeat washing fourtimes, discard the supernatants
  • carefully remove most of the supernatant
  • add 20µl of elution buffer, gently mix, wait a few minutes
  • briefly spin
  • carefully save the supernatant = eluate
  • add 20 µl of elution buffer, gently mix, wait a few minutes
  • add the supernatant to the eluat
  • add 40 µl Lämmli to the beads and eluat

analyse fractions by SDS-PAGE, load 5 µl per lane


Buffers, solutions, materials

Before starting the procedure decide which buffers you want to use. Please consider:
  1. type of buffer (Tris, Hepes, or phosphate)
  2. type of salt (Na+, K+, Mg2+ and concentration (from low 10 mM to high 1 M)
  3. detergent (Tween, SDS, Triton, RIPA....)
  4. DTT/beta-ME
Here are examples:

buffer for GSH beads:

Lysis buffer:
(1) PBS + additional 300 mM NaCl
(2) 50 mM Tris/HCl pH8, 500 mM NaCl, 1 mM DTT

Wash buffer:
(1) PBS+300 mM NaCl
(2) 50 mM Tris/HCl pH8, 500 mM NaCl, 1 mM DTT

Elution buffer
50 mM Tris/HCl pH8, 50 mM NaCl, 10 mM Glutathion
Prepare aliquots of 5 ml 100 mM Glutathion (pH 8) and freeze at -20C. Only shortly before using dilute these aliquots into 50 ml elution buffer. Check the pH. Do not store the 1x elution buffer.

buffer for Ni-NTA or Ni-Silica beads:

Lysis buffer:
(1) 50 mM Na-phosphate pH 8, 300 mM NaCl, 20 mM imidazol
(2) 50 mM Tris/HCl pH 8, 300 mM NaCl, 20 mM imidazol
When working with agarose beads the lysis buffer must contain 20 mM Imidazol to prevent unspecific binding. The Protino Ni-silica beads from Macherey-Nagel have a lower background binding. The lysis buffer only contains 1 mM Imidazol.

wash buffer
(1) 50 mM Na-phosphate pH 8, 300 mM NaCl, 20 mM Imidazol
(2) 50 mM Tris/HCl pH 8, 300 mM NaCl, 20 mM imidazol

elution buffer
50 mM Na-phosphate pH 8, 300 mM NaCl, 250 mM imidazol