Purification of LexA
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Inoculate 10 ml BL21(DE3) pET28a-His-LexA for O/N @ 37oC
Dilute 1:100 into 1 L LB+20 Kn & grow @ 37 oC, 1.5-3.5 hrs, 300 rpm until OD600=0.5.
Induce with 250 μM IPTG (500 μl of 0.5 M into 1 L) & grow 3 hr, 37 oC
Centrifuge in weighed 250 ml PP bottles at 8K/10’, discard supe and weigh pellet.
Freeze pellet @ -80 oC O/N
Pour 2 gels for each purification batch.
Transfer 4 ml of 50% slurry Ni-NTA beads to 50 ml tube column & wash 2X w/ 50 ml
Wash buffer.
Resuspend cells with at least 5 ml BugBuster / g pellet and transfer to 50 ml PP cent tube
Rock slowly in cold room 10-20 min.
Centrifuge 16,000 g/ 20 min /4 oC and transfer supernatant to 60 ml syringe with 45 μm
filter.
Collect filtrate in 50 ml tube; collect 10 λ whole cell lysate sample for gel.
Transfer washed beads to 50 ml tube; invert in cold room 15 min.
Pack beads in column and collect flow through in 50 ml tube
Apply 60 CV (120 ml) Wash buffer and collect in beaker.
Elute 2 ml fractions into 2 ml tubes as follows:
tube # CV
[Imidazole]
1-4
4
100 mM
5-7
3
250 mM
8-10 3
400 mM
11-12 2
750 mM
Samples boiled 5-10’, run 12% gel and Coomassie stain (see template below) as follows:
Tube #
MW Supe WCL
FT
W
fraction 1…10
beads
7.5 20 + 5λ 5X 20
20
20
20
+5
+5
+5
+5
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Pool LexA containing fractions with <90% and >50% purity and combine with fresh,
washed Ni-TA beads in 50 ml tube with 30 ml wash buffer overnight at 4 oC.
Repeat steps 13-16
Strip dialysis tubing with 1 mM EDTA and then rinse with water.
Dialyze against 1 L of LexA storage buffer including glycerol 3X, 3 hrs - O/N each time
Concentrate with 10K MWCO centricon using CH’s swinging bucket rotor @ 3900 g
Nanodrop (User: Lichtarge PW: t907) to determine OD280 and use  = 7300 M-1 cm-1 with
c = Abs / ( * l); LexA MW = 24389.8 g/mol
Alternatively, use BCA protein estimation kit but dilute so that glycerol is at 1%.
Wash buffer: 50 mM PO4 pH 6.8, 50 mM Imidazole, 1% glycerol, 500 mM NaCl pH 6.8
adjusted with phosphoric acid
5X Elution buffer: 250 mM PO4 pH 5.8-7.4? 5 M NaCl
Elution buffers: 50 mM PO4, 1 M NaCl, X mM imidazole pH 7.4 adjusted with phosphoric
acid
750 mM
5X Elution buffer
10.0 ml
1M imidazole
37.5 ml
H2O (q.s. 50 ml)
2.5 ml
LexA storage buffer: 10 mM PIPES-NaOH pH 7.0, 0.1 mM EDTA, 200 mM NaCl, 10%
glycerol
PIPES
9.1 g
0.5 M EDTA
600 λ
NaCl
35 g
Glycerol
300 ml
q.s. H2O
3L
pH 7.0 ~ 9 pellets NaOH & 10N NaOH dropwise if necessary
Notes:
Perform all steps at 4 oC if possible. Include glycerol in dialysis buffer.
If column gets clogged, can quick spin at 250 g but don’t allow bed to go dry.
Max cent. speed for beads is 700 g / 2’ (~2000 rpm in centrifuge across hall)
K156A mutant elution may precipitate on ice in presence of high imidazole
concentrations.
Do not leave protein elutions in >50 mM imidazole overnight.
Alternative to IPTG induction is autoinduction media from Studier ProtExpPur05
Load beads on plastic column by firmly attaching 25 ml pipette filled with lysate/bead
mixture by firmly pushing down and turning. Wrap junction with parafilm to prevent
leaks. Prior to attaching filled 25 ml pipette, fill interior of plastic column with
lysate/bead mixture to avoid a trapped air bubble in the column. Before collecting flow
through, allow column to pack by parafilming top air port of 25 ml pipette. In order to
add buffer to top of pipette, remove synthetic fiber from top with hooked wire and make
air port on the side of pipette (near the top but below the constriction) with a hot poker.
From Movahedzadeh, Davis Micro97)
This clone was transformed into E. coli BL21(DE3)pLysS. Cultures were grown in LB broth containing 200
lg ampicillin ml−" and 34 lg chloramphenicol ml−", and were induced at OD'!! 0±5 (1 cm pathlength,
Unicam UV2 spectrometer) by the addition of IPTG to a ®nal concentration of 0±1 mM (along with more
ampicillin to 200 lg ml−") and incubation continued for a further 3 h before harvesting.
Purification of M. tuberculosis LexA. The induced cells were harvested, resuspended in sonication
buffer (50 mM phosphate pH 8, 1 M NaCl), and lysed by sonication at 100 W for
4¬30 s. The LexA protein was puri®ed by means of the His tag introduced from the vector on a NiNTA (Qiagen) column. The lysate was applied at 1 ml min−", the column washed with
50 mM phosphate pH 5, 1 mM imidazole, 10% (w}v) glycerol, and the LexA protein eluted with 50
mM phosphate pH 8, 1 M NaCl, 0±25 M imidazole. The puri®ed protein was estimated by SDS-PAGE
to be "95% pure.