Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth Hormone |
| Abbreviated Name | GH |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | O14643 |
| 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 | 176 |
| Molecular Weight | 20450.3 |
| Pi | 8.36 |
| Molecular Weight | 20450.3 |
| Disulphides | Unknown |
| Full Sequence |
FPTIPLSRLFDNAMLRARRLYQLAYDTYQEFNPQTSLCFSESIPTPSNRVKTQQKSNLELLRISLLLIQSWLEPVQLLRSVFANSLVYGASDSNVYRHLKDLEEGIQTLMWRLEDGSPRTGQIFNQSYSKFDTKSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | St John RJ, Carpenter JF, Balny C, Randolph TW. (2001) Biologycal Chemistry, 276, 46856-63 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | High Pressure |
| Wash Buffer | n/a |
| Solubilization Buffer | n/a |
| Refolding Buffer | pH 6.0, 0.75 M GdnHCl, 1 mM EDTA, 0.1% sodium azide |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.0 |
| Refolding Temperature | 24.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Genentech Inc. generously supplied the recombinant human growth hormone (rhGH) for this study. rhGH was aggregated by rotating 10 ml of rhGH solution (1.75 mg/ml) in a 50-ml Falcon tube on a cell suspender for 24 h at 8 revolutions/min. Aggregation buffer consisted of 10 mM sodium citrate, 1 mM EDTA, 0.1% sodium azide, pH 6.0, at 24 °C (\"buffer\"). A second set of rhGH aggregates was prepared identically, with the addition of 0.75 M GdnHCl in the aggregation buffer. Prior to pressurization, aggregates were centrifuged at 13,000 × g for 15 min, and the supernatant was removed. The desired refolding buffer was then added, and the aggregates (0.87 mg/ml unless otherwise noted) were resuspended using a sonicating cell disrupter (Ultrasonics, 50% duty, 1-s cycles for 10 s). Sample Pressurization-- Pressure was generated using high pressure nitrogen (400 bar) connected to equipment from High Pressure Equipment Co. (Erie, PA) as previously described (26) or with high pressure crank generators from High Pressure Equipment Co. Samples were prepared either in heat-sealed bulbs of SAMCO transfer pipettes or in sterile, disposable syringes (one end heat-sealed and the other sealed with the rubber plunger). Sealed samples were placed into a 2-liter pressure vessel equipped with a heating jacket, rated to 2000 bar. Samples were slowly pressurized (15 min) to final desired pressure to minimize pressurization-induced heating (less than 2 °C as measured by a thermocouple mounted in the interior of the vessel lid). The depressurization rate was ~100 bar/min. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |