Refolding Record:
| Protein | |
|---|---|
| Protein Name | Macrophage inflammatory protein 1-beta |
| Abbreviated Name | MIP-1-beta |
| SCOP Family | interleukin 8-like chemokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P13236 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 67 |
| Molecular Weight | 7650.6 |
| Pi | 4.76 |
| Molecular Weight | 7650.6 |
| Disulphides | Unknown |
| Full Sequence |
MGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYDLELN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Laurence JS, LiWang AC, LiWang PJ. (1998) Biochemistry, 37, 9346-9354 |
| Project Aim | Structural Studies |
| Fusion | Thioredoxin |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 7 h |
| Expression Vector | pET32 |
| Expression Protocol | The amplified DNA was placed into the pET32LIC vector as instructed by the manufacturer and then sequenced. For each protein, the appropriate vector was transformed into BL21-(DE3) and an individual colony was subsequently grown in 1 L of minimal medium containing 15NH4Cl (Isotec, Mi-amisburg, OH) as the only nitrogen source. Cells were induced at A550 ) 0.9 by adding IPTG (Calbiochem, La Jolla, CA) to 1 mM, and the cells were harvested by centrifugation after about 7 h. The cell pellet was resuspended in 500 mM NaCl, 20 mM Tris, pH 8, 1 mM EDTA, 5 mM benzamidine, and 5 mM B-mercaptoethanol, passed through a French press two times at 16 000 psi, and then centrifuged at 19000g for 1 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.9 = 550 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 5 M guanidine hydrochloride, 50 mM NaCl, 20 mM Tris, pH 8, 1 mM EDTA, and 5 mM B-mercaptoethanol |
| Refolding Buffer | phosphate-buffered saline (PBS) pH 7.4 |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cell pellet was resuspended in 500 mM NaCl, 20 mM Tris, pH 8, 1 mM EDTA, 5 mM benzamidine, and 5 mM 􏰆-mercaptoethanol, passed through a French press two times at 16 000 psi, and then centrifuged at 19000g for 1 h. The resulting pellet was then resuspended in 15 mL of 5 M guanidine hydrochloride, 50 mM NaCl, 20 mM Tris, pH 8, 1 mM EDTA, and 5 mM 􏰆-mercaptoethanol (overall buffer adjusted to pH 8). The resuspended protein was centrifuged at 15 000g for 30 min to remove insoluble material and subjected to refolding (35). The refolded protein solution was loaded onto a Ni chelating column (Pharmacia, Piscataway, NJ) and eluted with midazole. |
| Refolding Assay | NMR analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |