Refolding Record:
| Protein | |
|---|---|
| Protein Name | Zona pellucida glycoprotein-C |
| Abbreviated Name | bmZPC |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bonnet Monkey |
| UniProt Accession | O19027 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 545 |
| Molecular Weight | 59269.1 |
| Pi | 7.95516 |
| Molecular Weight | 59269.1 |
| Disulphides | 0 |
| Full Sequence |
MRLLQCVLLCVSLSLVLSGQHKPETPGYSSVLHCGLWSFQFAVNLSQEATSPPVLITWDN
QGLLHKLQNDSDCGTWIRKRPGSSVVLEATYSSCYVTEWDSHYIMPVGVEGVGVAEHKMV
PERKLLKCPMDLLARDAPDTDWCDSIPARDRLPCAPSPISRGDCEGLGCCYSSENSCYYG
NTVTLRCTREGHFSIAVSRNVVSPPLLLDSVRLALRNDSACNPVMATQAFVLFHFPFTSC
GTTRRITGDRAVYENELVATRDVKNGSRGSVTRDSIFRLHVSCSYSVSSNSLPIKVQVFT
LPPPFPETQPGPLTLELQIAKDKNYGSYYGVGDYPVVKLLRDPIYVEVSILHRTDPSLGL
LLHQCWATPSTDPLSQPQWPILVKGCPYIGDNYQTQLIPVQKALDLPFPSHYQRFSIFTF
SFVDPTVEKQALRGPVHLHCSVSVCQPAETPSSVRTCPDLSRRRKFSTIFQNTTASVSSK
GPMILLQATKDPPEKLRAPVDSKVLWVAGLSGTLILGGLVVSYLAIKQLNCPDQTCQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Patra AK, Gahlay GK, Reddy BV, Gupta SK, Panda AK. (2000) Eur J Biochem., 267, 7075-7081 |
| 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 | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 2-4h |
| Expression Vector | pQE30 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 m m Tris/HCl buffer, pH 8.5, containing 5 m m EDTA and 2% deoxycholate |
| Solubilization Buffer | 100mM Tris-HCl, 2M urea, pH 12 |
| Refolding Buffer | 20 m m Tris buffer, containing 1 m m EDTA, 1 m m reduced glutathione, 0.1 m m oxidized glutathione and 10% sucrose |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Induced E. coli cells were lysed by French press at 124 MPa and inclusion bodies were recovered by centrifugation at 8000 g for 30 min at 4 °C. The inclusion body pellet obtained was washed with 50 m m Tris/HCl buffer, pH 8.5, containing 5 m m EDTA and 2% deoxycholate and the r-bmZPC inclusion bodies were purified as described previously [ 14]. The purified inclusion bodies were solubilized in 100 m m Tris/HCl buffer at different pH values (4-12). Solubilization of r-bmZPC from inclusion bodies was further optimized by adding different concentrations of urea (2-8 m). Fixed amounts of inclusion bodies (2 mg·mL 1) in the above buffers were left at room temperature for 30 min. After incubation, the suspensions were centrifuged at 8000 g for 10 min and the protein content of the supernatants and pellets were estimated using bicinchoninic acid assay (BCA) reagents (Pierce, IL, USA). Purified r-bmZPC inclusion bodies were solubilized in optimized conditions comprising 100 m m Tris buffer (pH 12) containing 2 m urea for 30 min at room temperature. The pH of the solubilized protein was brought down to 8.5 by adding 1 m HCl followed by extensive dialysis at 4 °C against renaturation buffer (20 m m Tris buffer, pH 8.5, containing 1 m m EDTA, 1 m m reduced glutathione, 0.1 m m oxidized glutathione and 10% sucrose). Refolded r-bmZPC was further purified using ion-exchange chromatography |
| Refolding Assay | Ligand Binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |