Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cystatin C |
| Abbreviated Name | Cystatin C |
| SCOP Family | Cystatins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01034 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 120 |
| Molecular Weight | 13347.1 |
| Pi | 8.74728 |
| Molecular Weight | 13347.1 |
| Disulphides | 2 |
| Full Sequence |
SSPG KPPRLVGGPM DASVEEEGVR RALDFAVGEY NKASNDMYHS RALQVVRARK QIVAGVNYFL DVELGRTTCT KTQPNLDNCP FHDQPHLKRK AFCSFQIYAV PWQGTMTLSK STCQDA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gerhartz B, Ekiel I, Abrahamson M. (1998) Biochemistry, 37, 17309-17317 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET3a-cystatin C |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | unknown |
| Solubilization Buffer | 6 M GdnHCl |
| Refolding Buffer | 50 mM ammonium bicarbonate |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | >30 mg/L |
| Refolding Time | at least 2h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Isolation and Refolding of Recombinant Cystatins. Purified inclusion bodies from 1 L cultures of expressioninduced bacteria were resolved in 5 mL of 6 M guadinium hydrochloride. The protein was refolded by SEC (Superdex 75 HR 10/30; Pharmacia Biotech) in 50 mM ammonium bicarbonate, pH 7.8. Protein Analyses. Analytical electrophoreses in agarose and SDS-polyacrylamide gels were performed with separation gels containing 15% acrylamide. Quantitative densitometric scanning of analytical gel electropherograms was performed using a Bio-Rad scanning system. The concentration of cystatin C was determined by A280 measurements. |
| Refolding Assay | Far-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 2 mg/L |
| Purity | |
| Notes | Wild-type, cytoplasmatic expression vector. NMR, ANS binding and fluorescence experiments were also carried out. |