Refolding Record:
| Protein | |
|---|---|
| Protein Name | Neuroserpin |
| Abbreviated Name | Neuroserpin |
| SCOP Family | Serpins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q99574 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Multi-domain proteins (alpha and beta) |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 394 |
| Molecular Weight | 44941.1 |
| Pi | 5.24343 |
| Molecular Weight | 44941.1 |
| Disulphides | 0 |
| Full Sequence |
MRGSHHHHHH ADLS VNMYNRLRAT GEDENILFSP
LSIALAMGMM ELGAQGSTQK EIRHSMGYDS LKNGEEFSFL KEFSNMVTAK ESQYVMKIAN SLFVQNGFHV NEEFLQMMKK YFNAAVNHVD FSQNVAVANY INKWVENNTN NLVKDLVSPR DFDAATYLAL INAVYFKGNW KSQFRPENTR TFSFTKDDES EVQIPMMYQQ GEFYYGEFSD GSNEAGGIYQ VLEIPYEGDE ISMMLVLSRQ EVPLATLEPL VKAQLVEEWA NSVKKQKVEV YLPRFTVEQE IDLKDVLKAL GITEIFIKDA NLTGLSDNKE IFLSKAIHKS FLEVNEEGSE AAAVSGMIAI SRMAVLYPQV IVDHPFFFLI RNRRTGTILF MGRVMHPETM NTSGHDFEEL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Belorgey D, Crowther DC, Mahadeva R, Lomas DA. (2002) J Biol Chem, 277, 17367-1737 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | SG13009 (pREP4) |
| Expression Temp | 30.0 |
| Expression Time | 3h |
| Expression Vector | pQE31 |
| Expression Protocol | Cells were grown in 2 liters of 2TY at 37degC until A600 reached 0.8-1.0; then 1mM IPTG was added, and the culture was transferred to 30 °C for a further 3 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.8-1.0 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 100mM NaCl, 10mM EDTA, 0.05% v/v Triton X-100, pH 7.8 |
| Solubilization Buffer | 10mM Tris-HCl, 100mM NaH2PO4, 6M guanidium chloride pH 8.0 |
| Refolding Buffer | 20 mM NaH2PO4, 100mM NaCl, pH 7.8 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 7.8 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 12h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were harvested by centrifugation, resuspended in wash buffer without Triton X-100 and disrupted by sonication. The cell pellet was washed three times with wash buffer and once again with wash buffer without Triton X-100, then dissolved in solubilization buffer. The protein was then refolded drop by drop overnight at 4degC in refolding buffer. The protein was then purified further using a HiTrap chelating column, followed by Q-sepharose. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |