Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ferritin light chain |
| Abbreviated Name | FLC |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P02792 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | L variant |
| Chain Length | 174 |
| Molecular Weight | 19888.5 |
| Pi | 5.51 |
| Molecular Weight | 19888.5 |
| Disulphides | Unknown |
| Full Sequence |
SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Levi S, Corsi B, Rovida E, Cozzi A, Santambrogio P, Albertini A, Arosio P. (1994) Biologycal Chemistry, 269, 30334-30339 |
| Project Aim | Purification & characterization |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pWMBLex2LFT |
| Expression Protocol | Ferritins and Variants-Human ferritin L-chain variants were obtained by oligonucleotide-directed mutagenesis of the plasmid pEMBLex2LFT described by Levi et al. (1992). Human H- and L-chain and variants were expressed in Escherichia coli and, when soluble, were purified as described by Levi et al. (1987,1992). Ferritins were electrophoretically pure. When needed they were treated to remove iron as described in Levi et al. (1988). Protein concentration was determined with BCA reagent (Pierce) using bovine serum albumin as a standard. Purification and Renaturation of the Insoluble L Variants-Some of the L variants accumulated only in the insoluble fraction of E. coli extracts even using different strains and different induction procedures. Renaturation studies of the insoluble ferritins were performed on precipitates from 20 g of cell paste. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | GdnHCI\' or urea |
| Refolding Buffer | 0.1 M sodium phosphate, pH 7.4, 3 mM DTT, 1 mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 3 mM |
| Refolding Protocol | They involved solubilization with GdnHCI\' or urea, and dilution followed by dialysis in renaturing buffers in the presence of 3 mM DTT, 1 mM EDTA. In method A, the precipitates were incubated in 60 ml of 6 M GdnHCI, pH 3.5, for 18 h, and after centrifugation the samples were diluted 10-fold into 0.1 M sodium phosphate, pH 7.4, at 4 \"C and then dialyzed against the same buffer. In method B the precipitates were resuspended in 20 ml of 2 M urea, pH 8.0,lOOO units of Benzonase (Merck) were added, and the samples were incubated at 37 \"C for 18 h to digest DNA and RNA. The variants were recovered by centrifugation, solubilized in 20 ml of 8 M urea, pH 3.0, at 4 \"C, diluted 10-fold into 0.1 M sodium phosphate, pH 7.4, and dialyzed at 4 \"C. |
| Refolding Assay | Protein Analyses |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |