Expression,
Purification
and
Crystallization
of
the
Mycobacterium Tuberculosis HSP16.3 Molecular Chaperone
Background of Mycobacterium Tuberculosis HSP16.3
HSP16.3, a 16.3 kDa protein from Mycobacterium Tuberculosis, was originally
identified as a prominent antigen (Kingston et al., 1987). During the stationary phase,
HSP16.3 is maximally expressed and becomes a main protein of the latent phase
(Yuan et al., 1996). Previous studies showed that HSP16.3 can make the cell structure
stable and prevent stationary Mycobacterium Tuberculosis from autolysing
(Cunningham et al., 1998). In previous studies, HSP16.3 was found as one of theα
-crystallin-related small heat shock proteins (sHSP) with molecular chaperone activity.
Experiments in vitro revealed that HSP16.3 can suppress the thermal aggregation of
citrate synthase at 39.5˚C, without consumption of ATP (Chang et al., 1996).
Now the Mycobacterium Tuberculosis HSP16.3 gene was cloned to the plasmid
pSTE-HSP16.3, and transformed to E.Coli. BL21(DE3) strain.
Material and Method
Expression
Things to have ready before Starting.
-Plate or glycerol culture
-Sterile LB 25ml in a 50mL shaker flasker, 250ml in a 500mL shaker flasker, all
together autoclaved, antibiotic added afterword.
- antibiotic and sterile water
- Tips
Prepare the LB and autoclave:
Fomula of the LB medium for 1 Liter:
Bacto Tryptone (BT)
10 g
Bacto Yeast Extract (BYE)
10 g
NaCl
10g
The LB medium, dd H2O and the tips all together autoclaved at 121 ˚C for 20
minutes.
Method:
1 Innoculate 25 ml LB Medium ( containing 100 ug) and grow culture overnight(37
˚C, 200rpm).
2 Next morning inoculate 250 ml prewarmed LB Medium ( containing 100 ug) with
the 25 ml overnight culture and grow at 37 ˚C, 200rpm, HSP16.3 was overexpressed
in soluble form intracellularly without IPTG induction.
3 Incubate the Culture for 10 hours before havesting the cell at 4000 g for 20 minutes.
4 Resuspend the cell pellet in 30 ml Butter A and freeze the Sample in -80˚C
refigerator.
Purification
DE52 Ion-Exchange column
Things to have ready before Starting.
-Butter A: 50 mM Imidazole pH 6.5 (1 liter)
-Butter B: 50 mM Imidazole pH 6.5 , 300mM NaCl
all together Fitrate with 0.2 um membrane.
- DE52 medium , column ,Gradient maker, UV-monitor and Fractioner
- Tips
Method:
1 Thaw the cell pellet and vortex .
2 Add 0.4ml 100 mM PMSF and sonicate (400kw, 4s-6s 50 cycle* 5 )
3 Centrifuge 15000 rpm, 30 minutes to pellet debris
4 Transfer supernatant to a 50 ml conicale tube and discard the pellet.
5 The supernatant dilute to 50 ml with Buffer A and then load to DE52 ion-exchange
columns (20ml), which was pre-equibrated with 100ml Buffer A. And then wash the
unbound proteins with 100 ml Buffer A.
6 Elute the protein with a linear gradient : 200ml buffer A plus 200ml buffer B,
2ml/min, 6ml each fraction.
7 Run 15% SDS-PAGE to determine the HSP16.3 peak.
Desalting by dialysis
1 Preparation of the dialysis tube
Cut the tube in a suitable length (20-30 cm)
Boil the tube in solution containing 10 mM NaHCO3 for a few minutes.
Boil the tube in solution containing 10 mM EDTA for a few minutes.
Rasin the tube with de-ion water
2 Pool the HSP16.3 peak and dialysis the Sample against 1000ml Buffer A for more
than 6hours.
Q-Separose (HP) Ion-Exchange Column
1 load the sample to Q-Separose (HP) Ion-Exchange column (20ml), which was
pre-equibrated with 100ml Buffer A. And then wash the unbound proteins with 100
ml Buffer A.
2 Elute the protein with a linear gradient : 200ml buffer A plus 200ml buffer B,
2ml/min, 6ml each fraction.
3 Run 15% SDS-PAGE to determine the purity of the HSP16.3 peak.
Gel filtration Column
The HSP peak was a final volumn 0.3ml and then run though a Superdex75 (HR,
10/30mm) gel filtration column in 150mM NaCl and 5mM Imdazole, pH6.5.
Crystallization
1 The purified HSP16.3 was solvent-exchanged to water and concentrated to 20mg/ml
before crystallization trails (Bradford). All the crystallization trials were carried out
using the hanging-drop vapor-diffusion method at 291K: drops consisted of 2
microlitres of HSP16.3 protein solution plus 2 microlitres of the precipitant. The
drops were equilibrated against 0.2 ml precipitant at room temperature. The
crystallization conditions were investigated with a PEG4000 Kit.
Result and discussion
The purity of the final HSP16.3 was over 95% by SDS-PAGE. The crystallization
trials of HSP16.3 yielded Cubic crystals with a size of 0.8*0.8*0.6mm in a few days.
de5206hsp01:1_UV1_280nm
de5206hsp01:1_Conc
mAU
Elution Profile of HSP16.3 on DE52 Column(25ml)
Buffer A:
50 mM Imidazole pH 6.5
Buffer B:
50 mM Imidazole pH 6.5 , 300mM NaCl
Flow Rate: 2ml/min
3500
3000
HSP
2500
2000
1500
1000
500
0
0
100
200
QHsp04:1_UV1_280nm
300
400
ml
QHsp04:1_Conc
mAU
600
HSP
500
400
300
200
100
0
0
100
200
300
400
ml
su1210:1_UV1_280nm
Elution Profile of HSP16.3 on Q-Separose
Column(High Performance, 25ml)
Buffer A:
50 mM Imidazole pH 6.5
Buffer B:
50 mM Imidazole pH 6.5 , 300mM NaCl
Flow Rate: 2ml/min
mAU
1200
HSP
1000
Elution Profile of HSP16.3 on Superdex200
800
(HR, 10/30mm)
Buffer: 5 mM Imidazole pH 6.5 , 150mM NaCl
Flow Rate: 0.75ml/min
600
400
200
0
0
20
40
60
80
100
ml
SDS-PAGE analysis of the purification of HSP16.3.
Lane1, molecular-weight markers (97.4 KD, 66.2KD,
45KD, 31KD, 14.4KD);
Lane 2, Fraction after Superdex 200 chromatography
(HR, 10mm/30cm);
Lane 3, Fraction after Q-Separose chromatography;
Lane 4, Fraction after DE52 cellulose chromatography.
Buffer
Tris-HCL
pH
8.5
Precipitant
PEG 4000
Method
Vapor Diffusion
Temperature
293 K
Size
0.8*0.8*0.6mm
References
Chang Z., Primm, T.P., Jakana J., Lee H. I., Serysheva I., Chiu W., Gilber H. F.,
Quiocho F. A., (1996) J Biol Chem 271:7218-7223
Cunningham A. F., Spreadbury C. L., (1998) J. Bacteriol. 184:801-808
Kingston A. E., Salgame P. R., Mitchison N.A., Colston M. J. (1987) Infect. Immun
55,3149-3154
Yuan Y., Crane D. D., Barry C. E. III (1996) J Bacteriol 178: 4484-4492