Refolding Record:
| Protein | |
|---|---|
| Protein Name | Tumor necrosis factor alpha |
| Abbreviated Name | TNF-alpha |
| SCOP Family | TNF-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01375 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Antibody single-chain fragment-TNF alpha chimera |
| Variants | n/a |
| Chain Length | 394 |
| Molecular Weight | 42696.9 |
| Pi | 8.7 |
| Molecular Weight | 42696.9 |
| Disulphides | Unknown |
| Full Sequence |
MARIQQSDAE LVKPGASVKI SCKASGYTFT DHAIHWAKQK PEQGLEWIGY ISPGNDDIKY NEKFKGKATL TADKSSSTAY MQLNSLTSED SAVYFCKRSY YGHWGQGTTL TGSGGGGSGG GGSGGGGSRI QMTQSPASLS VSVGELVTIT CRASENIYSN LAWYQQKQGK SPQLLVYAAT NLADGVPSRF SGSGSGTQYS LKINSLQSED FGSYYCQHFW GTPYTFGGGT KLEIKRV
VRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Yang J, Moyana T, Xiang J (1995) Molecular Immunology, 32, 873-881 |
| Project Aim | Drug Studies |
| 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 | 5h |
| Expression Vector | pT7-7 |
| Expression Protocol | Cells were grown in M9 media containing 2g/L LB broth base, 75microg/ml ampicillin, 20microg/ml chloramphenicol at 37degC until OD600 reached 0.6. Expression was induced with 1.0mM IPTG and cells were grown a further 5h before being harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Osmotic shock |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM TrisHCl pH 8.0, 20mM EDTA |
| Solubilization Buffer | 6M GdnHCl, 100mM TrisHCl pH 8.0, 2mM EDTA, 300mM DTT |
| Refolding Buffer | 100mM TrisHCl pH 8.0, 0.5M L-arginine, 8mM GSSG, 2mM EDTA |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | 0.03mg/ml |
| Refolding Time | 48h |
| Redox Agent | GSSG |
| Redox Agent Concentration | 8mM |
| Refolding Protocol | Harvested cells were resuspended in 50mM TrisHCl pH 8.0, 20mM EDTA, 0.25mg/ml lysozyme and incubated at 20degC for 1hr. 2% Triton X-100 and 0.5M NaCl were then added to the sample, which was incubated at 20degC for a further 30min. After centrifugation, the insoluble incluson bodies were resuspended in wash buffer and sonicated for 3min on ice. The inclusion bodies were then harvested and washed 3 times more. Inclusion bodies were dissolved in solubilization buffer to a final concentration of 3mg/ml and centrifuged. The supernatant was then diluted 100-fold with refolding buffer and incubated at 10degC for 48h. The sample was then concentrated by ultrafiltration and then dialysed against 20mM Na phosphate pH 6.8, 50mM NaCl and purified using a MonoQ column |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | 10mg/L culture |
| Purity | n/a |
| Notes | n/a |