Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rho-associated protein kinase 2 |
| Abbreviated Name | ROCK-2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | Q62868 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Catalytic domain of ROCK |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 548 |
| Molecular Weight | 62768.1 |
| Pi | 5.19 |
| Molecular Weight | 62768.1 |
| Disulphides | Unknown |
| Full Sequence |
PEAAAGDGAGAGRQRKLEALIRDPRSPINVESLLDGLNSLVLDLDFPALRKNKNIDNFLNRYEKIVKKIRGLQMKAEDYDVVKVIGRGAFGEVQLVRHKASQKVYAMKLLSKFEMIKRSDSAFFWEERDIMAFANSPWVVQLFCAFQDDRYLYMVMEYMPGGDLVNLMSNYDVPEKWAKFYTAEVVLALDAIHSMGLIHRDVKPDNMLLDKHGHLKLADFGTCMKMDETGMVHCDTAVGTPDYISPEVLKSQGGDGYYGRECDWWSVGVFLFEMLVGDTPFYADSLVGTYSKIMDHKNSLCFPEDTEISKHAKNLICAFLTDREVRLGRNGVEEIKSASFFKNDQWNWDNIRETAAPVVPELSSDIDSSNFDDIEDDKGDVETFPIPKAFVGNQLPFIGFTYFRENLLLSDSPPCRENDAIQTRKSEESQEIQKKLYALEEHLSSEVQAKEELEQKCKSINTRLEKTAKELEEEITFRKNVESTLRQLEREKALLQHKNAEYQRKADHEADKKRNLENDVNSLKDQLEDLKKRNQSSQISTEKVNQLQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Duan W, Wang S, Chen M, Wang C, Zhang L, Liu J, Sun L, Yan M. (2006) Biol Pharm Bull, 29, 38-42 |
| Project Aim | Recombinant Protein 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 |
| Expression Temp | 37.0 |
| Expression Time | 12 h |
| Expression Vector | pET28a |
| Expression Protocol | Positive BL(21) clones were cultured in LB culture media (tryptone 10g/l; yeast extraction 5g/l; 10g/l of NaCl) containing 30mg/ml kanamycin, at 37°C, 200rpm in a rocking incubator. When the absorbance (600nm, 1cm) of the culture media is about 0.8, different doses of IPTG were exposed to, and the temperature and rotation speed were adjust to room temperature and 100rpm respectively. After the clones were induced for 0—12h, the bacteria were isolated by centrifugation (4°C, 12000*g, 5 min). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20mMTrisCl, 0.2M NaCl, 2Murea, 1mM2-mercaptoethanol,pH 7.5 |
| Solubilization Buffer | 20mMTrisCl, 0.2M NaCl, 0.05Mimidazole, 6Mguanidine hydrochloride, 1mM2-mercaptoethanol, pH 7.5 |
| Refolding Buffer | 2ml with purified water |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 0.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Protein Extraction and Purification The ROCK-CD protein of rat (rROCK) was expressed as inclusion bodies, and the extraction was carried out as follow. Briefly, the bacteria from 100ml were suspensed in 4ml Suspense Buffer (20mMTrisHCl, pH 7.5), and was sonicated (300W, 10s sonication, 10s interval, 30 cycles) on ice. The precipitation was got by centrifugation (12000􏰂g, 4°C, 10min) and resuspensed in 4ml Resuspense Buffer (20mMTrisCl, 0.2M NaCl, 2Murea, 1mM2-mercaptoethanol, 2% Triton X-100, pH 7.5). The second sonication (300W, 10s sonication, 10s interval, 20 cycles) was carried out and the precipitation was obtained by centrifugation (12000*g, 4°C, 10min). Purified inclusion bodies were obtained by rinsing the precipitation twice with Resuspense Buffer without Triton X-100. The inclusion bodies were dissolved in 4ml Degeneration Buffer (20mMTrisCl, 0.2MNaCl, 0.05Mimidazole, 6Mguanidine hydrochloride, 1mM2-mercaptoethanol, pH 7.5) by vertex at room temperature for about 15min. After removed remaining particles by passing the sample through a 0.22mm filter, the purification procedure followed. HiTrap chelating 1ml column was washed with 5ml purified water using a 5ml syringe. Load 0.5ml 0.1MNiSO4and continue to wash with 5ml purified water. Equilibrate the column with 5ml Degeneration Buffer. Then Load the sample and wash the column with 10ml Degeneration Buffer and with 5ml Elution Buffer (20mMTrisCl, 0.2MNaCl, 0.5M imidazole, 1mM2-mercaptoethanol, pH 7.5). The Elution Buffer (containing rROCK-CD) was fractionally collected and stored at -20°C for detection and refolding. Refolding of rROCK-CD Let the Elution Buffer thawed at room temperature. rROCK-CD protein was separated by centrifugation (12000􏰂g, 4°C, 10min). Two milligrams precipitation was dissolved in 200ml Degeneration Buffer without imidazol. The refolding was accomplished by diluting the solution to about 2ml with purified water very slowly on ice. Guanidine hydrochloride was removed by ultrafiltration (10 kDa ultrafiltration tub, Millipore). rROCK-CD solution was adjusted to 1mg/ml (20mMTrisHCl, 20mMMgCl2, 1mM 2-mercaptoethanol, 0.56% NaN3, pH 7.2) and storedC. The protein concentration was determined by Coomassie brilliant blue assay kit (Nanjing Jiancheng Bio- engineering Institute, China). |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |