The Protein Purification
Facility
The Wolfson Centre for
Applied Structural Biology
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mariol@cc.huji.ac.il
Tel: 972-2-6586920 FAX: 972-2-6758963
Bacterial Expression Screen - DDM (Dodecyl Maltoside) lysis -
Small Affinity binding
Adapted from the talk of Dr.Nicola
Burgess-Brown (Structural Genomics Consortium,University of Oxford
Protein Production Workshop-
Oxford 6-7th July 2011
Screen for soluble or insoluble expression
1) Spin 1.5 ml aliquots of bacteria sample
5min 10,000rpm 4°C, discharge sup and keep pellet frozen -80°C
(Recommendation:
duplicate or triplicate for each sample to repeat experiment if necessary)
2) Disperse cells in 0.75 ml lysis buffer
and left in ice for ~20-30 minutes.
(Recommendation: use 1.3ml lysis
buffer for big pellet of ON overinduction
3) Froze cells with liquid nitrogen for less
than 20 minutes, and then thawed in room temperature water.
4) Spin lysates 15 minutes 14000 rpm at 4°C,
and separate sup and pellet. Disperse pellet in 0.75 ml of buffer. Keep a 40ul
sample of supernatant
for PAGE-SDS: soluble proteins; and 40ul sample of pellet: insoluble proteins or unlysed cells (Recommendation:
the use of resuspended pellet
instead of total cell sample, has the advantage
that you can see if the cells were lysed or not more efficiently)
5) Run samples in PAGE-SDS
for Coommasie stain (total proteins) and Western using anti His-HRP
for detection (specific protein).
Run samples in pairs of sup-pellet.
Buffer: Tris 50 mM
pH 7.5, 10% glycerol, 0.5M NaCl
Lysis buffer: Buffer
+ 0.1% dodecyl maltoside + 1 mM PMSF + 0.2 mg/ml lysozyme + 50μg/ml DNAase (prepare
fresh!!!)
Small
Screen for IMAC binding
6) Equilibration of IMAC resin: place 50ul beads (100ul suspension) of
Ni-NTA agarose beads (or any other commercial beads) in 1.5ml plastic tube.
Wash with 1 x 1.5ml H2O and 2 x 1.5ml buffer (washing: mix,
spin 3min 3500rpm, and remove supernatant).
7) Mix the supernatant of last step gently with the equilibrated resin for
60 min at 4°C
8) Spin 3min 3500rpm 4°C. Remove supernatant and keep sample of
40µl for PAGE-SDS: unbound proteins
9) Wash beads with 1ml buffer several times (washing: mix, spin 3min
3500rpm, keep supernatant aside, be careful not to take the resin) up to
OD280nm <0.05 or at least three times. Keep sample of 40µl for PAGE-
SDS
of each
washing.
10) Elute recombinant protein with 2x250ul buffer + 250mM Imidazol
(incubate 3 to 5min each time before spinning 3min, 3500rpm at 4°C).
Keep
sample of 40ul for PAGE-SDS of each elution.
Keep the rest of the sample to check the oligomeric state (aggregation) of the
protein by
analytical gel filtration (GF)
11) Resuspend beads in 50ul H2O
+ 20ul 5x sample buffer. Mix and spin. Keep sample of 40ul for PAGE-SDS: SDS extracted beads.
12) According to the number of screening
samples, run on PAGE-
SDS:
A)
Crude supernatant; resuspended
pellet; unbound, and first elution
B)
Crude supernatant; resuspended pellet;
unbound, washings, elutions, and SDS
extracted beads.
C)
Only first elution
13) Check oligomeric state (aggregation) of the protein by analytical GF
the relevant samples
Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for
Applied Structural Biology, The Hebrew University of Jerusalem
mariol@cc.huji.ac.il
Tel: 972-2-6586920 FAX: 972-2-6758963
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©, 2002, The Hebrew University of Jerusalem. All Rights Reserved.