Refolding Record:
| Protein | |
|---|---|
| Protein Name | Trichosanthes kirilowii defensin |
| Abbreviated Name | TDEF1 |
| SCOP Family | Plant defensins |
| Structure Notes | |
| Organism | Trichosanthes kirilowii |
| UniProt Accession | Q19JA1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 48 |
| Molecular Weight | 5613.6 |
| Pi | 9.57 |
| Molecular Weight | 5613.6 |
| Disulphides | Unknown |
| Full Sequence |
RTCQRASQTWKGVCFRNEKCRNNCLREKART
GNCKYVFRACICYFPC
|
| Notes | It is known there are 2-6 disulfide bridges to stabilize the structure. |
| Expression | |
|---|---|
| Report | Da-Hui L, Gui-Liang J, Ying-Tao Z, Tie-Min A. (2007) Appl Microbiol Biotechnol, 74, 146-151 |
| Project Aim | Recombinant Protein Expression |
| Fusion | N-terminal thioredoxin + hexahistidine tag |
| 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 | 3 h |
| Expression Vector | pET32a(+) |
| Expression Protocol | Construction of the vector pET32a(+)-TDEF1 To obtain clones merely with the mature peptide coding region of TDEF1, a primer, called MP1 with BamHI site (5′-CGA GGA  TCC AGA ACA TGT CAG-3′), was designed based on the mature peptide coding sequence of the target clone obtained above. PCR reaction (with MP1 and SP2 as primers) were as follows: 94°C for 30 s, 55°C for 45 s, 72°C for 30 s, and finally, 72°C for a 10-min extension. The amplified product was cloned into pMD18-T and sequenced. The recombinant vector pMD18-T-TDEF1 was digested with BamHI and HindIII, and the resulting fragment of TDEF1 was cloned into the BamHI and HindIII sites of the expression vector pET32a(+), which was designated pET32a(+)-TDEF1. It was introduced into E. coli BL21 (DE3) cells. The empty vector was also introduced into BL21 (DE3) as a control. Fusion protein production in E. coli E. coli BL21 (DE3) cells containing a plasmid of interest were grown at 37°C for 14 h in 5 ml LB medium containing appropriate antibiotics (50 μg/ml of ampicillin). The culture was diluted 1:100 (v/v) into 300 ml of the same medium, followed by shaking at 37°C until an OD600 of 0.5 was reached. Isopropyl β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM, and cells were grown at 37°C for 3 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM Tris-HCl |
| Solubilization Buffer | 8 M urea (containing 50 mM Tris–HCl buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM dithiothreitol (DTT), pH 8.0) |
| Refolding Buffer | 50 mM Tris–HCl buffer, 3 mM glutathione reduced, 1 mM glutathione oxidized, pH 8.0), containing 6, 4, 2, 1, and 0.5 M urea |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | DTT/PMSF |
| Redox Agent Concentration | 10 mM/1 mM,10 mM/1 mM |
| Refolding Protocol | Recombinant bacteria induced by IPTG were collected by centrifugation and suspended in 50 mM Tris–HCl buffer (pH 8.0). Then, the bacteria cell lysate was prepared by sonication at 4°C. The supernatant was removed by centrifugation at 12,000×g for 15 min, and the pellet (inclusion bodies) was used for purification. The inclusion bodies were dissolved in 8 M urea (containing 50 mM Tris–HCl buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM dithiothreitol (DTT), pH 8.0), and centrifuged at 12,000×g for 15 min to collect the supernatant for 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. The protein refolding was processed by dialysis against the buffer systems (50 mM Tris–HCl buffer, 3 mM glutathione reduced, 1 mM glutathione oxidized, pH 8.0), containing 6, 4, 2, 1, and 0.5 M urea, respectively, to decrease, gradually, the concentration of urea in the protein solution to 0.5 M, then kept it overnight at 4°C, and finally, the residual urea was eliminated by dialysis against the same buffer but containing no urea. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |