Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prolactin |
| Abbreviated Name | Prolactin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01236 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 201 |
| Molecular Weight | 22898.2 |
| Pi | 6.14 |
| Molecular Weight | 22898.2 |
| Disulphides | 3 |
| Full Sequence |
LPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMNQKDFLSLIVSILRSWNEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDSHKIDNYLKLLKCRIIHNNNC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Langenheim JF, Tan D, Walker AM, Chen WY. (2006) Mol Endocrinol., 20, 661-674 |
| 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 | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | PET22b |
| Expression Protocol | Chemically competent Rosetta (DE3) pLysS E. coli cells (Novagen) were transformed with the constructs and propagated overnight at 37 C with agitation in Luria-Bertani broth containing 100 μg/ml ampicillin. The next morning 40 ml of each of the starter cultures was used to inoculate 1 liter of Terrific Broth in baffled flasks, and the cultures were grown to an OD600 of 0.9 before induction of protein production with 1 mm isopropyl-β-thiogalactoside (Alexis Biochemicals, San Diego, CA). After 4 h of induction the cells were harvested by centrifugation at 5000 × g for 5 min and resuspended in solution 1 (20 mm Tris·HCl; 10 mm EDTA; 0.5% Triton X-100; pH adjusted to 8.0) to which 0.4 mg/ml lysozyme was added. The cells were incubated at room temperature for 1 h to weaken the cell membranes before the cells were sheared on ice with five 1-min ultrasonic pulses (30% duty at setting 7) using a 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.9 = 600 |
| Cell Disruption Method | ultrasonic pulses |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mm Tris·HCl; 10 mm EDTA; 1 m urea; pH adjusted to 8.0 |
| Solubilization Buffer | 20 mm Tris··HCl; 10 mm EDTA; 8 m urea; 1% vol/vol β-mercapto-ethanol; pH adjusted to 8.0) |
| Refolding Buffer | 20 mm Tris·HCl; 10 mm EDTA, pH 8.0 containing decreasing amounts of urea (4 m urea, 2 m urea, 0 m urea, 0 m urea) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The insoluble fraction containing the inclusion bodies was recovered by centrifugation at 12,000 × g for 30 min at 4 C and resuspended in solution 2 (20 mm Tris·HCl; 10 mm EDTA; 1 m urea; pH adjusted to 8.0). The inclusion bodies were collected by centrifugation at 12,000 × g for 30 min at 4 C and were solubilized and denatured in solution 3 (20 mm Tris··HCl; 10 mm EDTA; 8 m urea; 1% vol/vol β-mercapto-ethanol; pH adjusted to 8.0). The proteins were refolded by dialysis against baths of 20 mm Tris·HCl; 10 mm EDTA, pH 8.0 containing decreasing amounts of urea (4 m urea, 2 m urea, 0 m urea, 0 m urea). The refolded proteins were filtered through 0.45 μm filters and purified by anion-exchange chromatography using a 5 ml HiTrap Q Sepharose HP column (Amersham Biosciences, Piscataway, NJ) on an ÄKTA fast protein liquid chromatography system (Amersham Biosciences). The concentration of the purified proteins was determined using the Coomassie Plus Protein Assay Reagent (Pierce Chemical Co., Rockford, IL). The purified proteins were separated by nonreducing and reducing SDS-PAGE along with BSA standards (Pierce) and stained with SYPRO Orange (Molecular Probes, Eugene, OR) to confirm their concentration and purity. The identity of the proteins was confirmed by Western blotting. |
| Refolding Assay | Western Blot,Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 95% |
| Purity | n/a |
| Notes | n/a |