Refolding Record:
| Protein | |
|---|---|
| Protein Name | Metalloproteinase inhibitor 1 also known as Tissue inhibitor of metalloproteinas |
| Abbreviated Name | TIMP-1 |
| SCOP Family | Tissue inhibitor of metalloproteinases, TIMP |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01033 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 185 |
| Molecular Weight | 20708.8 |
| Pi | 8.46 |
| Molecular Weight | 20708.8 |
| Disulphides | Unknown |
| Full Sequence |
CTCVPPHPQTAFCNSDLVIRAKFVGTPEVNQTTLYQRYEIKMTKMYKGFQALGDAADIRFVYTPAMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWNSLSLAQRRGFTKTYTVGCEECTVFPCLSIPCKLQSGTHCLWTDQLLQGSEKGFQSRHLACLPREPGLCTWQSLRSQIA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kleine T, Bartsch S, Bläser J, Schnierer S, Triebel S, Valentin M, Gote T, Tschesche H. (1993) Biochemistry, 32, 14125-14131 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HMS174DE3 |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET11a |
| Expression Protocol | Expression of rTIMP-I. For expression of rTIMP-1, the E. coli strain HMS 174 [DE31 was transformed with PET-1 la-TIMP-1 to allow T7 RNA polymerase mediated transcription of the cloned TIMP-1 cDNA. A culture of HMS 174 [DE31 [PET-lla-TIMP-11 was grown overnight at 37 C in medium containing 10 g of Bacto tryptone, 5 g of yeast extract, and 10 g of NaCl per liter and 100 Fg/mL ampicillin. Two liters of the same medium were inoculated with 20 mL of the overnight culture at 37 “C. When a cell density corresponding to an OD578 of 0.6 was reached, IPTG was added to a final concentration of 0.4 mM, and the incubation was continued for another 4 h at 37 “C to maximize the production of rTIMP- 1. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6 = 578 |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris-HC1 (pH 7.5) and 6 M urea |
| Solubilization Buffer | 50 mM Tris-HC1 (pH 8.5), 8 M urea, 1 mM EDTA, and 200 mM 8-mercaptoethanol. |
| Refolding Buffer | 100 mM Tris-HC1 (pH 8.7), 100 mM NaC1, 5 mM CaC12, and O.Ol% TritonX-100 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.7 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 60 h |
| Redox Agent | GSSG |
| Redox Agent Concentration | 80 mM,80 mM |
| Refolding Protocol | Purification and Refoldingof rTIMP-I. A 15-mL aliquot of the frozen crude extract was thawed and loaded onto a Sephacryl S-200 column (1000 mL) previously equilibrated with equilibration buffer containing 20 mM Tris-HC1 (pH 6.9), 4 M urea, 10 mM DTE, 200 mM NaC1,l mM EDTA, and 0.005% Triton X-100. Equilibration and elution were performed at a flow rate of 7.5 mL/h. Fractions were assayed for rTIMP- 1 by Western immunoblotting of SDS-polyacrylamide gels, as previously described (Blaser et al., 1991), and those containing rTIMP- 1 were pooled and dialyzed against reduction buffer (100 mM Tris-HC1 (pH 73,150 mM DTE, and 4 M urea). Protein content was determined in the dialysate by the Bradford method (Bradford, 1976). Oxidation buffer (100 mM Tris-HC1 (pH 7.9, 80 mM oxidized glutathione, and 4 M urea) was added to this dialysate, so that a final rTIMP-1 concentration of 20 pg/mL was reached. To reduce the concentration of the denaturing and reducing reagents and to initiate refolding, this mixture was diluted at 4 C with dilution buffer (100 mM Tris-HC1 (pH 8.7), 100 mM NaC1, 5 mM CaC12,andO.Ol%TritonX-100) to a final concentration 0.3 M urea. This dilution step was performed by adding 6 mL/h dilution buffer while gently stirring, thus allowing reshuffling of the mismatched disulfide bonds. This reaction mixture was then concentrated 15-fold in an Amicon ultrafiltration device equipped with a YM 5 membrane. After concentration, the buffer was exchanged against TIMP-buffer (20 mM Tris-HC1 (pH 7.9, 200 mM NaCl, 5 mM CaC12, and 0.005% Triton X- 100) by dialysis. The inhibitory activity of rTIMP-1 was analyzed by a modified method of Masui et al. (1977), and the concentration of rTIMP-1 was measured by TIMP-1 ELISA. |
| Refolding Assay | Kinetic analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 80% |
| Purity | n/a |
| Notes | n/a |