Refolding Record:
| Protein | |
|---|---|
| Protein Name | tumour necrosis factor alpha single-chain variable fragment |
| Abbreviated Name | TNF-scFv |
| SCOP Family | Immunoglobulin light chain kappa variable domain |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | A2KBC4 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 309 |
| Molecular Weight | 33923.0 |
| Pi | 8.47 |
| Molecular Weight | 33923.0 |
| Disulphides | 2 |
| Full Sequence |
QIQLVQSGPELKKPGETVKISCKASGYTFTHYGMNWVKQAPGKG LKWMGWINTYTGEPTYADDFKEHFAFSLETSASTVFLQINNLKNEDTATYFCARERGDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCTASQSVSNDVVWYQQKPGQSPKMLMYSAFNRYTGVPDRFTGRGYGTDFTFTISSVQAEDLAVYFCQQDYNSPRTFGGGT
KLEIKQIPELENNEKAPKVVILKKETAYILSVQAEEQQKLISEEDLLRKRREQLKHKLEQLRNSCA
hhhhhh
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Liu M, Wang X, Yin C, Zhang Z, Lin Q, Zhen Y, Huang H (2006) Biotechnol. Appl. Biochem., 43, 137-145 |
| Project Aim | Protein refolding |
| Fusion | C-terminal myc tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)Star |
| Expression Temp | 37.0 |
| Expression Time | 5h |
| Expression Vector | pTCM |
| Expression Protocol | The transformed cells [E. coli BL21(DE3) star] harbouring the expression vector pTS were grown in 500 ml of LB (Luria–Bertani) broth medium supplemented with kanamycin (50 mg/ml) at 37 °C. When the cultures reached an attenuance (D600) value of 0.8, IPTG was added at a final concentration of 0.4 mM to induce the target gene expression. After 5 h of induction, the cells were harvested by centrifugation. A 1 g (wet weight) portion of cells from the 500 ml culture was resuspended in 200 ml of buffer A (50mM Tris/HCl(pH 8.0), 100mM NaCl, 2mM EDTA, 100ug.ml lysozyme and 10ug.ml DNase) and then treated by sonication at 4 °C for 30 min. The sonicated material was centrifuged at 12000 rev./min at 4 °C for 20 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 800 = 0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 20mM Tris/HCl (pH 8.0), 3% Triton X-100, 1M Urea |
| Solubilization Buffer | 10mM Tris/HCl (pH 8.0). 8M Urea, 0.1M NaH2PO4, 10mM BME |
| Refolding Buffer | 20mM Tris/HCl (pH 8.0), 100mM NaCl, 4mM GSSG/4mM GSH, 5mM beta-cyclodextrin |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 4mM,4mM |
| Refolding Protocol | Inclusion body pellets obtained from the above treatment were washed with 200 ml of buffer B (20mM Tris/HCl (pH 8.0)3% Triton X-100, 1M Urea) and then 200 ml of buffer C (20mM Trix/HCl (pH 8.0)). During each wash, the pellets were resuspended in each wash buffer (buffers B and C) and then stored at 25 °C with gentle shaking for 2 h, followed by a centrifugation at 11000 g for 20 min. After being washed, the pellets were resuspended in 100 ml of denaturing buffer (buffer D (10mM Tris/HCl (pH 8.0), 8M Urea, 0.1M NaH2PO4, 10mM BME) plus 10 mM imidazole and stored in 25 °C overnight with gentle shaking to ensure that the inclusion bodies were entirely dissolved. The mixture was centrifuged at 14000 g at 4 °C for 20 min and the supernatant was then loaded on to a 5 ml Ni-NTA–agarose column which was pre-equilibrated with denaturing buffer (buffer D) supplemented with 10 mM imidazole at a flow rate of 0.5 ml/min. On-column purification and refolding were performed as follows by several changes of buffers. First, the column was washed with denaturing buffer (buffer D) supplemented with 20 mM imidazole to remove non-specifically bound contaminants. In the next step, the column was washed with 10 CV (column volumes) of buffer E (20mM Tris/HCl(pH 8.0), 100mM NaCl, 0.1% Triton X-100, 4mM GSSG/4mM GSH) containing 0.1% Triton X-100 at a flow rate of 1 ml/min. This was followed by a wash with 10 CV of buffer F (containing 5 mM b-cyclodextrin at a flow rate of 3 ml/min to remove detergent from the (20mM Tris/HCl(pH 8.0), 100mM NaCl, 0.1% Triton X-100, 4mM GSSG/4mM GSH, 5mM beta-cyclodextrin) detergent–protein complexes and to allow the protein to fold. Before elution, additional wash with buffer C containing 0.5 M NaCl was applied to remove remaining impurities and b-cyclodextrin. Refolded TNF-scFv protein was eluted with buffer C that was supplemented with 300 mM imidazole. This was followed by a 0.5 M NaOH wash to remove the small amount of unfolded protein that aggregated on the column. The total eluted TNF-scFv protein was dialysed against 2000 ml of 20 mM Tris/HCl buffer (pH 8.0) for 6 h at 4 °C to remove the residual imidazole and impurities, followed by centrifugation at 12000 rev./min for 30 min, and was then filtered through a 0.22-mm-pore-size filter to remove potential aggregations. The fraction flowing through the column, the fractions washed out respectively by 20 mM imidazole, by Triton X-100 and by b-cyclodextrin, and the fraction eluted by 300 mM imidazole during the chromatographic procedure were collected and analysed by SDS/PAGE. The purity of the eluted TNF-scFv protein was determined by using the gel scanning quantitative analysis. The final product was quantified with the Bradford method by using BSA as the control and the refolding yield was calculated. |
| Refolding Assay | Native PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Triton X-100 |
| Additives Concentration | 0.1% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |