Refolding Record:
| Protein | |
|---|---|
| Protein Name | Gamma-crystallin E |
| Abbreviated Name | Cryge |
| SCOP Family | Crystallins/Ca-binding development proteins |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | CRGE_RAT |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 175 |
| Molecular Weight | 21132.6 |
| Pi | 7.24 |
| Molecular Weight | 21132.6 |
| Disulphides | Unknown |
| Full Sequence |
GKITFYEDRGFQGRHYECSTDHSNLQPYFSRCNSVRVDSGCWMLYEQPNFTGCQYFLRRGDYPDYQQWMGFSDSVRSCRLIPHSSSHRIRIYEREDYRGQMVEITDDCPHLQDRFHFSDFHSFHVMEGYWVLYEMPNYRGRQYLLRPGEYRRYHDWGAMNARVGSLRRIMDFY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Goode D, Crabbe MJ. (1994) Archives Biochemistry and Biophysics, 315, 104-110 |
| Project Aim | Cloning and expression |
| 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 | 12-14 h |
| Expression Vector | pET3d |
| Expression Protocol | Induction of Rat gamma-E Crystallin Expression Cultures of transformed BL21(DE3) were inculated into Luria/ampicillin broth (either at 37c with vigorous shaking or 25c with slow shaking), grown to late log phase, and then induced to express crystallin by addition of IPTG to a fnal concentration of 0.4 mM. Incubation was continued for 12-14 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | Guanidine-Hcl 8M, DTT 10 mM, BRJ35 detergent 0.01% |
| Refolding Buffer | Tris–HCl 50 mM (pH 8.4), EDTA 1 mM BRJ35 0.01%, DTT 10 mM |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.4 |
| Refolding Temperature | 30.0 |
| Protein Concentration | 1.4 mg/ml |
| Refolding Time | overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | 10 mM,10 mM |
| Refolding Protocol | Inclusion bodies were purified by the method of Sambrook et al. (19). Partialy purified inclusion bodies contaning expressed gamma E crystallin were solubilized in Guanidine-Hcl 8M, DTT 10 mM, BRJ35 detergent 0.01% or urea (8M)/ NaCl (0.1 M) containing DTT 10 mM pH adjusted to 2 with HCl, and the protein concentration was adjusted to 1.4 mg/ml. The unfolded gamma-crystallin solution was placed in a Hamilton Microlab M automatic pipeting system set to deliver successive 10-ul aliquots at 20-s intervals (1.8 ml/h). Aliquots of unfolded protein were delivered overnight into a 1-liter vol of refolding bufer under either nonreducing conditions Tris–HCl 50 mM (pH 8.4), EDTA 1 mM BRJ35 0.01% or reducing conditions where DTT (10mM) was added to the buffer. this was maintained at 30 c in a water-jacketed vessel containing an electrical stirrer. |
| Refolding Assay | Circular Dichroism (uv-CD),Tryptophan Fluorescence |
| Refolding Chaperones | None |
| Refolding Additives | Brij 35 |
| Additives Concentration | 0.01% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |