Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human macrophage elastase catalytic domain |
| Abbreviated Name | hMECD |
| SCOP Family | Matrix metalloproteases, catalytic domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P39900 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | catalytic domain |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 366 |
| Molecular Weight | 42123.6 |
| Pi | 8.56 |
| Molecular Weight | 42123.6 |
| Disulphides | 1 |
| Full Sequence |
GPVWRKHYITYRINNYTPDMNREDVDYAIRKAFQVWSNVTPLKFSKINTGMADILVVFARGAHGDFHAFDGKGGILAHAFGPGSGIGGDAHFDEDEFWTTHSGGTNLFLTAVHEIGHSLGLGHSSDPKAVMFPTYKYVDINTFRLSADDIRGIQSLYGDPKENQRLPNPDNSEPALCDPNLSFDAVTTVGNKIFF
FKDRFFWLKVSERPKTSVNLISSLWPTLPSGIEAAYEIEARNQVFLFKDDKYWLISNLRPEPNYPKSIHSFGFPNFVKKIDAAVFNPRFYRTYFFVDNQYWRYDERRQMMDPGYPKLITKNFQGIGPKIDAVFYSKNKYYYFFQGSNQFEYDFLLQRITKTLKSNSWFGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cheng DH, Shen Q, Qian J, Qian Z, Ye QZ. (2002) Acta Pharmacol Sin, 23, 143-151 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pGEMEX-hMECD |
| Expression Protocol | BL21 (DE3) pLYyS cells containing the plasmid pGEMEX-hMECD was cultured in 1 L of TY medium in the presence of ampiciin ( 100 mg/L) with shaking at 37 c to a cell density at OD600 of 0.6. Four hour after induction with IPTG 1 mmol/L, cells were harvested by centrifugation for 5 min at 8000 x g at 4 c. The cells were washed with Tris HCl buffer 50 mmol/L pH 7.5 and lysed by sonication at 4 c. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Tris HCl 50 mM pH 7.5,buffer containing urea 2 mol/L |
| Solubilization Buffer | 8 mM urea |
| Refolding Buffer | Tris HCl 50 mM pH 7.5 , zinc & calcium ions |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The pellets were washed three times by suspension in Tris HCl 50 mmol/L pH 7.5 followed by centrifugation. Then the pellets containing inclusion bodies were washed with Tris HCl 50 mmol/L pH 7.5 buffer containing urea 2 mol/L. Finally the pellets were solubilized in urea 2 mol/L. Finally the pellets were solubilized in urea 8 mmol/L. The concentration of denaturing agent in the solubilized inclusion bodies was lowered from 8 to 6 mol/L and clarified by centrifugation ( 15000 x g,20 min) and supernatant fluid was collected for purification. The urea extract 6 mol/L containing the hMECD protein was loaded on to a Hi-Performance Q-Sepharose column (20 mL) equilibrated previously with Tris HCl 50 mmol/L-urea 6 mol/L pH 7.8 and eluted with a linear NaCl gradient (0-1 mol/L) in the Tris HCl 50 mmol/L-urea 6 mol/L pH 7.8, and fractions containing the hMECD protein were collected. The fraction added slowly drop wise to a refolding buffer containing Tris HCl 50 mmol/L pH 7.5 and zinc and calcium ions. The mixture was centrifuged (15000 x g 20 min) to remove insoluble protein and then concentrated by using Amicon 8200 stirred cell with a 10000 NMWL Amicon PM 10 membrane for ultrafiltration. |
| Refolding Assay | Enzyme activity,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 23 mg |
| Purity | n/a |
| Notes | n/a |