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.    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