Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prolactin |
| Abbreviated Name | Prolactin |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Trichosurus vulpecula (Brush-tailed possum) |
| UniProt Accession | O62781 |
| 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 | 199 |
| Molecular Weight | 22928.0 |
| Pi | 5.53 |
| Molecular Weight | 22928.0 |
| Disulphides | 3 |
| Full Sequence |
LPICPSGAVNCQVSLSDLFDRAVMLSHYIHSPSSEMFNEFDERYAQGRGFITKAINSCHTSSLSTPEDKEQAQQIHHEDLLNLILRVLRSWNDPLYHLVTEVRSMQEAPDTILSKAMEIEEQNKRLLEGMEKIVGQVHPGDRENEVYSVWSGLPSLQMADEDTRLFAFYNLLHCLRRDSHKIDNYLKLLKCRLIHDSNC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Crawford JL, Lun S, Demmer J, Eckery DC. (2005) General and Comparative Endocrinology, 142, 297-307 |
| Project Aim | Undefined |
| 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 | 3 h |
| Expression Vector | pET23 |
| Expression Protocol | Expression, purification, and re-folding of recombinant possum Prl Single colonies of BL21 (DE3) pLysS bacteria transformed with pET23/posPrl were cultured in 3 mL Luria broth containing ampicillin (200 μg/mL) overnight at 37 °C using a modified version of a previously described method (Chen et al., 1998). Then, an aliquot of culture (2 mL) was diluted 25 times in the same medium and incubated at 37 °C with agitation until the OD600 nm reached 0.5. Recombinant protein expression was then induced by incubating for 3 h with isopropyl-β-D-thiogalactoside (final concentration 0.4 mM) and cells were pelleted by centrifugation at 3000g for 10 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM Tris–HCl, pH 7.5 |
| Solubilization Buffer | 8 M urea, 1% β-mercaptoethanol in 0.2 M sodium phosphate (pH 7.0) |
| Refolding Buffer | 50 mM NH4HCO3 with eight changes over 3 days at 4 °C |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 3 day |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | The pellet containing the bacteria was resuspended in 15 mL of ice-cold wash buffer (50 mM Tris–HCl, pH 7.5) and stored at −80 °C for protein purification. The bacterial pellet was thawed and after addition of protease inhibitors (P8465; Sigma Chemical, St. Louis, MO, USA), cells were sonicated on ice until the solution cleared. Inclusion bodies containing the expressed recombinant protein were pelleted by centrifugation (10,000g for 10 min at 4 °C) and washed with 15 mL wash buffer before the final centrifugation (10,000g for 10 min at 4 °C) step. Inclusion bodies were denatured by 1 h incubation in 8 M urea, 1% β-mercaptoethanol in 0.2 M sodium phosphate (pH 7.0) at room temperature and the resulting solution was dialysed against 50 mM NH4HCO3 with eight changes over 3 days at 4 °C. The concentration of recombinant posPrl expressed was determined using the BCA Protein Assay Reagent Kit (Pierce Chemical) following the manufacturer’s instructions. The extent of protein re-folding was determined by SDS–PAGE of aliquots denatured in the presence or absence of a reducing agent (2-mercaptoethanol). Analytical reverse phase high performance liquid chromatography (HPLC) was used to further purify the recombinant posPrl before it was freeze-dried for storage at −20 °C. |
| Refolding Assay | HPLC,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The refolding pH is not stated in this paper |