Refolding Record:
| Protein | |
|---|---|
| Protein Name | Chymotrypsin inhibitor |
| Abbreviated Name | NACI |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | chinese cobra Nanja atra |
| UniProt Accession | Q5ZPJ7 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 57 |
| Molecular Weight | 6391.1 |
| Pi | 8.31 |
| Molecular Weight | 6391.1 |
| Disulphides | Unknown |
| Full Sequence |
RPRFCELAPSAGSCFAFVPSYYYNQYSNTCHSFTYSGCGGNANRFRTIDECNRTCVG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Cheng YC, Yan FJ, Chang LS. (2004) Biochemica et Biophysica Acta, 1747, 213-220 |
| Project Aim | Structural Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET29a |
| Expression Protocol | Expression of NACI Synthetic oligonucleotides were designed to produce an amplified DNA fragment spanning the open reading frame of NACI. The forward primer introduced a 5′-EcoRV site preceding Arg-1 of NACI: 5′-GATATCCGTCCAAGGTTCTGTGAACTGGCT, with the reverse primer being 5′-GGTCATCCAACACAGGTGCGGTT-3′ (PI-down). The PCR product was cloned into pGEM-T easy vector. The inserted DNA fragment was cut with EcoRV and EcoRI, then ligated into the large fragment of EcoRV/EcoRI-cut pET-29a(+). The resulting plasmid was transformed into E. coli strain BL21(DE3). Transformants were selected on LB-agar plates supplemented with 50 ug/ml ampicillin. For the purpose of gene expression, E. coli BL21(DE3) cells harboring the plasmid were grown at 37 °C in LB medium containing 50 ug/ml ampicillin. After OD550 reached 1.0, isopropyl-B-d-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM. The culture was induced for periods of up to 4 h. The cells were harvested and lysed using ultrasonication. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1.0 = 550 |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10 mM Tris–1 mM EDTA (pH 8.0) containing 0.5% Triton X-100 |
| Solubilization Buffer | 10 ml of 50 mM sodium borate (pH 8.5) containing 5 mM EDTA, 8 M urea, and 4 mM reduced and 2 mM oxidized glutathione |
| Refolding Buffer | 10 ml of 50 mM sodium borate (pH 8.5) containing 5 mM EDTA, and 4 mM reduced and 2 mM oxidized glutathione |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 1 day |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,4mM/2mM,4mM/2mM |
| Refolding Protocol | The recombinant NACI was found to appear exclusively in the inclusion bodies of E. coli. The inclusion bodies recovered from 1 l of bacterial culture were thoroughly washed with 10 mM Tris–1 mM EDTA (pH 8.0) containing 0.5% Triton X-100, and sulfonated with disodium 2-nitro-5-(sulfothio)-benzoate (NTSB) in 8 M urea, 0.3 M Na2SO3, pH 8.5 [23]. The protein was precipitated with 1% acetic acid and dissolved in 10 ml of 50 mM sodium borate (pH 8.5) containing 5 mM EDTA, 8 M urea, and 4 mM reduced and 2 mM oxidized glutathione. Refolding was performed by a fourfold dilution with the same buffer without urea. After standing for 1 day at room temperature, the refolded protease inhibitor was further purified by HPLC on a SynChropak RP-P column (4.6 mm×25 cm) and eluted with a linear gradient of 7.5%–75% acetonitrile for 50 min. The flow rate was 0.8 ml/min, and the effluent was monitored at 280 nm. The pET29a(+) possesses a thrombin site for cleavage of the fusion protein, thus the purified protein was hydrolyzed with thrombin at 37 °C for 20 h. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |