Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glucagon-like peptide 1 receptor |
| Abbreviated Name | GLP-1 receptor |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P43220 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | N-terminal Extracellular domain aa.1-125 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 147 |
| Molecular Weight | 17244.2 |
| Pi | 8.79673 |
| Molecular Weight | 17244.2 |
| Disulphides | 3 |
| Full Sequence |
MRGSHHHHHHGS AGPRPQGATV SLWETVQKWR EYRRQCQRSL
TEDPPPATDL FCNRTFDEYA CWPDGEPGSF VNVSCPWYLP WASSVPQGHV YRFCTAEGLW LQKDNSSLPW RDLSECEESK RGERSSPEEQ LLFLY GRKRKRKRKN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bazarsuren A, Grauschopf U, Wozny M, Reusch D, Hoffmann E, Schaefer W, Panzner S, Rudolph R (2002) Biophysical Chemistry, 96, 305-318 |
| Project Aim | Structure-Function |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15(pREP4) |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pQE30 |
| Expression Protocol | Cells were grown on minimal medium, the growth rate was restricted by constant feeding with glucose and protein expression was induced with 0.4mM IPTG when OD600 reached 50. Cells were incubated a further 4h then were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 50 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl, 0.1M Tris, 1mM EDTA, 100mM DTT, pH 8.0 |
| Refolding Buffer | 100mM Tris pH 8.5, 500mM L-arginine, 1mM GSH, 5mM GSSG, 1mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 10.0 |
| Protein Concentration | |
| Refolding Time | 36h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/5mM |
| Refolding Protocol | Following cell lysis (details not provided) and isolation of inclusion bodies, the inclusion bodies were dissolved in 50ml solubilization buffer for 2h at room termperature at a protein concentration of 10mg/ml. The pH was adjusted to 4 with HCl and the mixture was centrifuged (20000rpm x 15min). The supernatant was dialyzed twice against 1L of 4M GdnHCl, pH 3.0 to remove DTT. For refolding of the protein, a folding reactor was used - 6 x 5ml pulses of solubilized protein were injected in intervals of 6h into a vessel containing 500ml of refolding buffer. The stirrer was set to 400rpm and 10degC. After 36h the refolding solution was centrifuged and diluted with 1.5L of 20mM Tris, 1mM EDTA pH 7.4 (Buffer A) to form a precipitate which was removed by centrifugation (1hr x 10000rpm). The protein was then loaded onto 20ml SP-Sepharose Fast Flow resin equilibrated in Buffer A and binding was allowed for 30min. The resin was then poured into a column, washed with Buffer A and the protein was eluted with 1M NaCl in the same buffer. The eluted protein was then diluted 10-fold into Buffer A and applied to a Hi-Trap SP-Sepharose column which was then washed with Buffer A. The protein was eluted using a salt gradient from 150mM to 1M NaCl. Finally the protein was loaded onto a Superdex 75 column in 20mM Tris, 1mM EDTA pH 7.4. |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | |
| Purity | |
| Notes | |