Refolding Record:
| Protein | |
|---|---|
| Protein Name | C-phycoerythrin class I alpha chain |
| Abbreviated Name | CpeA |
| SCOP Family | Phycobilisome |
| Structure Notes | |
| Organism | Synechococcus sp. (strain WH8102) |
| UniProt Accession | Q7U4Q1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 167 |
| Molecular Weight | 17817.1 |
| Pi | 5.45 |
| Molecular Weight | 17817.1 |
| Disulphides | Unknown |
| Full Sequence |
MKSVVTTVVT AADAAGRFPS QNDLEAVQGN IQRAAARLEA AEKLAAGLDN VTREAGDACF
NKYAYLKQPG EAGDSQVKID KCYRDLGHYL RLINYCLIVG GTGPLDEWGI AGAREVYRTL
GLPTNAYIEA LTYTRDRACA PRDMSAQALN EFKSYLDYAI NALS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Swietnicki, W. (2006) Curr. Opin. Biotech., 17, 367-372 |
| Project Aim | Structure-Function,Structural Studies,Assembly,Recombinant Protein Expression,Protein refolding |
| Fusion | N-terminal hexahistidine tag + thrombin cleavage site |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | Bl21 |
| Expression Temp | 23.0 |
| Expression Time | 3 |
| Expression Vector | pETDuet-1 |
| Expression Protocol | standard protocol |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 50 mM PBS, 0.5 M NaCl, pH 7.2 |
| Solubilization Buffer | 8 M Urea, 50 mM PBS, 0.5 M NaCl, pH 7.2 |
| Refolding Buffer | 50 mM PBS, 0.1 M TRIS, 0.15 M NaCl, 1 mM EDTA, 1 mM ß-Mercaptoethanol, pH 7.2 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no |
| Refolding pH | 7.2 |
| Refolding Temperature | 23.0 |
| Protein Concentration | n/a |
| Refolding Time | 3 h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1 mM,1 mM,1 mM |
| Refolding Protocol | gelfitration on Superdex200pg 16/60, flow 0.75 ml/min, room temperature, sample volume: 3 ml in 8 M Urea gelfiltration results in three peaks, indicating complex formation of protein |
| Refolding Assay | UV spectrum/Near UV CD/ Immunology |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 80 |
| Purity | 95 |
| Notes | all fractions of gelfiltration show typical CD-spectra of alpha-helical proteins |