Refolding Record:
| Protein | |
|---|---|
| Protein Name | Growth Hormone Receptor |
| Abbreviated Name | GHR |
| SCOP Family | Fibronectin type III |
| Structure Notes | |
| Organism | Oncorhynchus masou (Cherry salmon) (Masu salmon) |
| UniProt Accession | Q90W21 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | extracellular domain o ms GHR |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 222 |
| Molecular Weight | 26066.4 |
| Pi | 5.17 |
| Molecular Weight | 26066.4 |
| Disulphides | 2 |
| Full Sequence |
EPPTSEQALPQIRPQITGCVSHDMNTFRCRWNVGTFQNLTEPRDLRMFYYINNKNISPKEWSECPNYMADRIDECFFNESYTKVWITYSVQLRSGDQDNLYDEVIFTVEEIVEPDPPIALNWTLLNVGLTGSHFDIMVSWEPPHSADVSMGWMTLQYEVQYREVNSTLWRMVNLEKGRQRSLYGLRTNTDHEVRVRCKTLASRNFGEFSDPIVIHIPTKESR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fukada H, Ozaki Y, Pierce AL, Adachi S, Yamauchi K, Hara A, Swanson P, Dickhoff WW. (2004) General and Comparative Endocrinology, 139, 61-71 |
| Project Aim | Cloning and expression |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pCR-T7 |
| Expression Protocol | The fragment encoding the extracellular domain of msGHR was amplified with PCR using the full-length msGHR as a template. The primers for histidine-msGHR extracellular domain (msGHR-ECD) were as follows; msGHR-ECD (F): 5′-GAG CCG CCA ACT TCT GAA CAA GCC-3′ and msGHR-ECD(R): 5′-TCG GGA CTC TTT AGT GGG GAT G-3′. The PCR was done using proof reading Taq polymerase (Advantage 2 polymerase, Clontech). The PCR products were cloned to pCR-T7 vector (Invitrogen) and transfected to Top-10F′ E. coli cells. The vectors from positive clones were purified and transformed into BL21(DE3) pLysS E. coli strain for protein expression. Transfected cells were grown in 100 ml of LB-broth medium containing 20 μg/ml ampicillin at 37 °C in a 250 ml-flask. When the absorbance at 600 nm reached 0.4, isopropylthiol-β-D-galactoside was added to the tube at a final concentration of 0.4 mM. Cells were grown for an additional 4 h, and then collected by centrifugation at 5000g for 15 min and frozen at −30 °C. Inclusion bodies were purified using BugBuster Protein Extraction Reagent (Novagen). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 ml of 4.5 M urea with 40 mM Tris base (pH 11.0),Cysteine 0.1 mM,1 mM phenylmethanesulfonyl fluoride (PMSF |
| Refolding Buffer | Dilution with 2 vol. of 0.5% Tween-60 in water and dialysis with 1 × 5 L of 10 mM Tris–HCl (pH 8.0),All buffers contained 1 mM phenylmethanesulfonyl fluoride (PMSF) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 48 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | nclusion bodies were purified using BugBuster Protein Extraction Reagent (Novagen). Refolding was done using a modified method of Bignon et al. (1994). Purified inclusion bodies (from 100 ml culture volume) were dissolved in 20 ml of 4.5 M urea with 40 mM Tris base (pH 11.0). Cysteine was added to 0.1 mM and allowed to stand for 1 h at room temperature. The solution was diluted with 2 vol. of 0.5% Tween-60 in water, and then dialyzed for 2 days with 1 × 5 L of 10 mM Tris–HCl (pH 8.0). All buffers contained 1 mM phenylmethanesulfonyl fluoride (PMSF) to prevent protein degradation. Refolded proteins were purified by Ni–NTA column chromatography (Novagen) according to the manufacturer’s instruction. To purify the monomer form of the protein, purified fractions were subjected to ion exchange chromatography using a DEAE-52 (Whatman, Maidstone, Kent, UK) column (2 × 10 cm) equilibrated with 10 mM Tris–HCl (pH 8.0). Elution was carried out at room temperature using a step gradient of NaCl (0, 0.1, 0.2, and 0.4 M) in the same buffer at a flow rate of 60 ml/h. Protein concentration in fractions was determined by a BCA protein assay kit (Pierce, Rockford, IL, USA) using bovine serum albumin (BSA) as a standard. |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | Tween 60 |
| Additives Concentration | 0.5% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |