O-Separose(HP) lon-Exchange column I 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 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 cond itions were investigated with a PEg4000 Kit Result and discussion The purity of the final HSP16. 3 was over 95% by SDS-PAGE. The crystall ization trials of HSP16.3 yielded Cubic crystals with a size of 0.8*0.8*0.6mm in a few daysQ-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