Refolding Record:
| Protein | |
|---|---|
| Protein Name | Annexin B1 |
| Abbreviated Name | AnnB1 |
| SCOP Family | Annexin |
| Structure Notes | |
| Organism | Cysticercus cellulosae |
| UniProt Accession | Q9XZL9 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | Annexin B1-urokinase |
| Variants | n/a |
| Chain Length | 615 |
| Molecular Weight | 68213.8 |
| Pi | 7.81 |
| Molecular Weight | 68213.8 |
| Disulphides | 6 |
| Full Sequence |
MAYCRSLVHL YAPNGEKYKP TITPTPGFSP TADAEHLKRA MRGLGTNERA IIDILGNRTS AERMAIRDAY PSISSKTLHD ALTSELSGKF RRFALLLIQS PWQVMAEALY DAMKGAGTKE RVLNEIIAGC SKDDIPQLKK AFEEVSGGET LDDAIKGDTS GDYREALLLA LAGQADEPQA MQLKNLTPST LSQVVNPGLA ETDAKELYAC GEGRPGTAES RFMRPIVNRS FLQLNATNEA YNRAYGHPLI DAIKKETSRD LEDFLITRVR YATDRASLFA ELLHFAMRGA GTKDSTLQRV LALRADTDLG SIKEKYAELY GETLEAAIKG DTSGDYEALC LKLIGPA
LKFQCGQ KTLRPRFKII GGEFTTIENQ PWFAAIYRRH RGGSVTYVCG GSLMSPCWVI SATHCFIDYP KKEDYIVYLG RSRLNSNTQG EMKFEVENLI LHKDYSADTL AHHNDIALLK IRSKEGRCAQ PSRTIQTICL PSMYNDPQFG TSCEITGFGK ENSTDYLYPE QLKMTVVKLI SHRECQQPHY YGSEVTTKML CAADPQWKTD SCQGDSGGPL VCSLQGRMTL TGIVSWGRGC ALKDKPGVYT RVSHFLPWIR SHTKEENGLA L
|
| Notes | Urokinase chimeric partner: aa.144-411 of mature urokinase (aa. 164-431 of precursor) UniProt: UROK_HUMAN SCOP family: Eukaryotic proteases SCOP Unique ID:50543 |
| Expression | |
|---|---|
| Report | Yan H, Wang, W, He, Y, Zhao Z, Gao Y, Zhang Y, Sun S (2004) Acta Biochimica et Biophysica Sinica, 36, 184-190 |
| 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-RIL |
| Expression Temp | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET28a |
| Expression Protocol | Cells were grown in 2% LB broth (pH 7.4) at 37degC. When A600 reached 0.6, 0.5mM IPTG was added and growth continued for a further 3h. The final A600 was about 1.2 |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 1:8M urea, 0.5M NH4Cl, 20mM TrisHCl pH 8.5, 10mM DTT 2:8M urea, 0.1M NH4Cl, 20mM TrisHCl pH 8.5, 5mM DTT |
| Refolding Buffer | 1.5mM urea, 0.4mM L-arginine, 0.5mM EDTA, 10g/L glycerol, 0.2mM GSSG, 0.05M GSH, 20mM TrisHCl pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG/DTT |
| Redox Agent Concentration | 0.05/0.2/5-10mM |
| Refolding Protocol | 5g of inclusion bodies was suspended in 75ml solubilization buffer 1 and shaken at 4h at room temperature. After centrifugation, the supernatant was dialysed against solubilization buffer 2 for 12h, then dropped slowly into 800ml refolding buffer. The sample was then dialyzed against 10mM Gly/NaOH pH 9.0 with stirring for 24h at 4degC. During dialysis, the buffer was exchanged about 3 times. Insoluble materials were then removed by centrifugation and the supernatant was stored for further purification (DEAE-sepharose, Superdex-200) |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Glycerol |
| Additives Concentration | 1%/.4mM |
| Refolding Yield | 20mg/g IB |
| Purity | 95% |
| Notes | n/a |