Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human Gelatinase A |
| Abbreviated Name | GaCDfn |
| SCOP Family | Matrix metalloproteases, catalytic domain |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P08253 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 552 |
| Molecular Weight | 62059.4 |
| Pi | 5.01 |
| Molecular Weight | 62059.4 |
| Disulphides | 7 |
| Full Sequence |
YNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLRFSRIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTGVGGDSHFDDDELWTLGEGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLVAAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPDIDLGTGPTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTVTPRDKPMGPLLVATFWPELPEKIDAVYEAPQEEKAVFFAGNEYWIYSASTLERGYPKPLTSLGLPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMDP
GFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKLENQSLKSVKFGSIKSDWLGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cheng D, Shen Q, Nan F, Qian Z, Ye QZ. (2003) Protein Expression and Purification, 27, 63-74 |
| Project Aim | Identification and Characterization |
| 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-GaCD fn |
| Expression Protocol | BL21(DE3)pLysS cells containing the plasmid pGEMEX-GaCDfn or pGEMEX-GaCD were cultured in 2 L TY medium in the presence of ampicillin (100 ug/ml) with shaking at 37 °C up to a cell density at OD600 of 0.6 before induction of expression by adding isopropyl B--thiogalactopyranoside to 1 mM. Cells were harvested after 4 h by centrifugation for 5 min at 6000g. The cells were washed with Tris–HCl buffer (50 mM, pH 7.5) and lysed by sonication. The pellets were washed three times by suspension in the Tris buffer, followed by centrifugation (15,000g, 20 min). |
| 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 |
| Solubilization Buffer | Tris-Hcl 50 mM, 8 M urea |
| Refolding Buffer | Tris–HCl (50 mM, pH 7.5), CaCl2 (10 mM), and ZnCl2 (10 μM) |
| 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 |
| Refolding Protocol | Then, the pellets containing inclusion bodies were washed with the Tris buffer containing 2 M urea (20 ml). Finally, the recombinant protein was solubilized in 5 ml Tris buffer containing 8 M urea. The mixture was clarified by centrifugation (15,000g, 20 min) and the supernatant was collected and diluted to 6 M urea for purification. The denatured GaCDfn or GaCD proteins in the Tris/urea buffer were loaded onto a Q-Sepharose column (20 ml) equilibrated previously with buffer A (50 mM Tris–HCl, pH 7.8, 6 M urea) and eluted with a linear NaCl gradient (0–1 M) in the buffer A, and fractions containing the GaCDfn or GaCD protein were collected and stored at 4 °C. Protein samples taken throughout the purification procedure were analyzed by 12% reducing SDS–PAGE gel and the protein content of eluted fractions was estimated using the Bradford method with bovine serum albumin as the standard [26]. For investigating the effect of zinc ion, calcium ion, ionic potential or Brij-35 on GaCDfn refolding, we equalized the concentration of each variable in reaction mixtures for excluding the effect of it on GaCDfn activity, and thus, we could study each of them on GaCDfn refolding alone. The purified GaCDfn was refolded directly by a 10-fold dilution into a refolding buffer containing Tris–HCl (50 mM, pH 7.5), CaCl2 (10 mM), and ZnCl2 (10 μM), while purified GaCD was refolded by dialysis against a simple Tris buffer (50 mM, pH 7.5) without any added metal ions. The mixtures of GaCDfn were centrifuged (15,000g, 20 min) to remove insoluble proteins and concentrated by using an Amicon 8200 stirred cell (Millipore, Bedford, MA, USA) with an Amicon PM10 ultrafiltration membrane (MWCO 10 kDa). The concentrated GaCDfn was purified further on Hi-Performance Q-Sepharose (Uppsala, Sweden). Metal-free GaCDfn was prepared by dialysis (MWCO 10 kDa) against a Tris buffer (50 mM, pH 7.5) at 4 °C with four buffer changes. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 21% |
| Purity | n/a |
| Notes | n/a |