Refolding Record:
| Protein | |
|---|---|
| Protein Name | Chemotaxis inhibitory protein |
| Abbreviated Name | CHIPS |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Staphylococcus aureus |
| UniProt Accession | A6QIG7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | CHIPS 31-113 |
| Chain Length | 83 |
| Molecular Weight | 9726.1 |
| Pi | 9.1 |
| Molecular Weight | 9726.1 |
| Disulphides | Unknown |
| Full Sequence |
FEPFPTNEEIESNKKMLEKEKAYKESFKNSGLPTTLGKLDERLRNYLKKGTKNSAQFEKMVILTENKGYYTVYLNTPLAEDRK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gustafsson E, Forsberg C, Haraldsson K, Lindman S, Ljung L, Furebring C. (2008) Protein Expression and Purification, 1, 1 |
| Project Aim | Purification & characterization |
| Fusion | N-terminal +C terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | BL21star(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pAB128/CHIPS31–113 |
| Expression Protocol | Escherichia coli BL21star(DE3)pLysS were transformed with plasmids pAB128/ADC-1004 and pAB128/CHIPS31–113 and grown on LB agar plates containing ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml). One colony per CHIPS variant was inoculated into 10 ml LB supplemented with carbenicillin (100 μg/ml) and chloramphenicol (34 μg/ml) and cultured overnight at 30 °C and 200 rpm. Then the overnight cultures were centrifuged at 4500 rpm and the pellets were resuspended in new LB containing carbenicillin (100 μg/ml). Four milliliter of this was transferred into 200 ml LB with carbenicillin (100 μg/ml) and grown at 37 °C at 200 rpm in 1 l baffled glass flasks or at 300 rpm in 500 ml Ultra Yield™ flasks with AirOTop seals (Thomson Instrument Company, Oceanside, CA). The cultures were grown until OD600 reached 0.6–0.8 at which time isopropyl β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. Prior to IPTG induction, one milliliter of culture was taken out and pelleted in a microcentrifuge and was referred to as the uninduced sample. The cultures were harvested after 3 h by centrifugation at 4500 rpm for 15 min. one milliliter samples for SDS–PAGE were pelleted separately in a microcentrifuge. Pellets were stored at −20 °C until analysis on SDS–PAGE or isolation of inclusion bodies. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | deoxycholate buffer |
| Solubilization Buffer | 0.8 ml 50 mM Tris–HCl, 0.2 M NaCl, 2 mM EDTA, 7 M GuHCl pH 8.0 |
| Refolding Buffer | PBS supplemented with 0.5 M l-Arginine |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 0.0 |
| Refolding Temperature | 22.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were purified with washing in deoxycholate buffer, in a protocol modified from [7] and [8]. The following buffers were used: Buffer 1: 50 mM Tris pH 8.0, 1 mM EDTA, 25% Sucrose, buffer 2: 20 mM Tris pH 8.0, 0.2 M NaCl, 0.5% Sodium Deoxycholate, 2 mM EDTA and buffer 3: 10 mM Tris pH 8.0, 0.25% Sodium Deoxycholate, 1 mM EDTA. 100 ml culture pellets were thawed and resuspended in 5 ml of buffer 1. The suspension was sonicated to homogeneity on ice with two 30 s pulses, and inclusion bodies were pelleted by centrifugation at 12,000 rpm. The pellets were then resuspended in 5 ml of buffer 2 and sonicated to homogeneity on ice, followed by incubation with shaking at RT for 30 min, and centrifugation at 12,000 rpm. Finally, pellets were resuspended in buffer 3 and sonicated to homogeneity on ice, and inclusion bodies were pelleted by centrifugation at 12,000 rpm. Pellets were then kept at −20 °C until solubilized and refolded.Pellets were solubilized in 0.8 ml 50 mM Tris–HCl, 0.2 M NaCl, 2 mM EDTA, 7 M GuHCl pH 8.0 by incubation with shaking at RT for 10 min. Any remaining insoluble material was removed by centrifugation at 16,000g for 15 min. The supernatants were filtered through a 0.45 μm membrane and then refolded by drop-wise dilution at RT into PBS supplemented with 0.5 M l-Arginine to a final protein concentration of max 100 μg/ml with vigorous stirring. The protein solutions were then further incubated with stirring at RT over night. The following day, the solutions were centrifuged at 4500 rpm and filtered through a 0.45 μm membrane before concentration to a final volume of 1.2 ml on AmiconUltra spin filters (Millipore, Billerica, MA). |
| Refolding Assay | Circular Dichroism (uv-CD),Biological assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5 M |
| Refolding Yield | 62% |
| Purity | 80% |
| Notes | Refolding pH not stated |