Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth/differentiation factor 5 |
| Abbreviated Name | GDF5 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P43026 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 121 |
| Molecular Weight | 13581.6 |
| Pi | 6.46 |
| Molecular Weight | 13581.6 |
| Disulphides | Unknown |
| Full Sequence |
APLATRQGKRPSKNLKARCSRKALHVNFKDMGWDDWIIAPLEYEAFHCEGLCEFPLRSHLEPTNHAVIQTLMNSMDPESTPPTCCVPTRLSPISILFIDSANNVVYKQYEDMVVESCGCR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Honda J, Andou H, Mannen T, Sugimoto S. (2000) J Biosci Bioeng., 89, 582-9 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | W3110M |
| Expression Temp | 37.0 |
| Expression Time | 24 h |
| Expression Vector | pKK223-3 |
| Expression Protocol | Briefly, plasmid pKOT 245 of 3.7 kb m pKK223-3 (Pharmacia Biotech) with the insertion of a DNA fragment coding rhGDF5 between the tat promoter and rrnBTiT* terminator, and the E. coli strain W3110M (K12 origin) was transformed with this plasmid. This E. coli was cultivated in a 15001 large-scale bioreactor (Chiyoda Corp., Yokohama) containing 700 1 media (final volume) consisting of Bacto Tryptone (Difco, NJ, USA), citric acid, glucose, phosphates, various metals and thiamine HCl. rhGDF5 was induced with 1 mM of isopropyl-B-D-thiogalactopyranoside in the early log phase. The reagents were obtained from Wako (Osaka), Nacalai (Kyoto) or Dojin (Kuma- moto). After 24 h of fed-batch cultivation, E. coli was harvested using a continuous centrifuge (Westfalia Separator, Oelde, Germany) at 12,000 xg, washed with 25 mM Tris-HCl buffer (pH 7.3) containing 10mM ethylenediamine tetraacetic acid (EDTA), and disrupted using a high-pressure homogenizer (APV Gaulin, West Sussex, UK) operated at 560 bars (SOOOpsi) and passed through three times. The inclusion body was isolated using the same continuous centrifuge and washed with 20 mM Tris-HCl buffer (pH 8.3) containing 1 M urea and 5 mM EDTA. The productivity of rhGDF5 was about 5 g per 1 media, and after isolation, approximately 40 kg of wet inclusion body was obtained, which is equivalent to approximately 3 kg of rhGDF5. The inclusion body was then stored in infusion bags in a freezer (-SO’C) for subsequent use. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM Tris-HCl buffer (pH 8.3) containing 1 M urea and 5 mM EDTA. |
| Solubilization Buffer | 50mM glycine (pH 8.9 with NaOH), 8 M urea, 32 mM cysteine HCI and 5 mM EDTA. |
| Refolding Buffer | 0.5 M argenin-NaOH (pH 8.9), 0.5 NaCl, 20 mM CHAPS 4.8 mM cysteine HCl, 2.4 M urea, 0.75 mM EDTA |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.9 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 20 h |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 4.8 mM |
| Refolding Protocol | Direct refolding In the optimized laboratory-scale direct refolding protocol, solubilization was performed by mixing thawed inclusion body (100 g wet weight) with the following reagents and pure water, and adjusting the mixture to a final volume of 300 ml at a protein concentration of 16 mg. ml-l at room temperature. The reagents were 50mM glycine (pH 8.9 with NaOH), 8 M urea, 32 mM cysteine HCI and 5 mM EDTA. This solubilized inclusion body solution was diluted to 2 I using a refolding buffer with reagents pre-dissolved to give the final compositions listed in Table la. This solution was incubated at 4°C without stirring for approximately 20 h until the dimerization was complete. In the pilot-scale direct refolding trial, this was scaled up by a factor of 70, with a final refolding volume of 140 1, and incubated by mild stirring (30 rpm). |
| Refolding Assay | Protein activity assay,Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,CHAPS |
| Additives Concentration | 20 mM/0.5 M |
| Refolding Yield | 63% |
| Purity | n/a |
| Notes | n/a |