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| Refolding Record [Created: 2008-12-08 Updated: 2008-12-08 ] |
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PROTEIN |
| Protein Name |
Snakin-1 |
| Abbreviated Name |
SN1 |
| SCOP Family |
Unknown |
| Structure Notes |
n/a |
| Organism |
Solanum tuberosum (Potato) |
| UniProt Accession |
Q948Z4 |
| SCOP Unique ID |
n/a |
| Structure Solved |
|
| Class |
Unknown |
| Molecularity |
Unknown |
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CONSTRUCT |
| Full Length |
y |
| Domain |
n/a |
| Chimera |
n/a |
| Variants |
n/a |
| Chain Length |
63 |
| Molecular Weight |
6907.1 |
| pI |
8.97 |
| Disulphides |
Unknown |
Pfam Domain(s) and
Number of Occurrences |
Pfam Entry
GASA(1) |
| Domain Graphics |
 |
| Full Sequence |
GSSFCDSKCKLRCSKAGLADRCLKYCGICCEECKCVPSGTYGNKHECPCYRDKKNSKGKSKCP
BLAST against Refold database | BLAST against NCBI NR database
|
| Notes |
n/a |
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EXPRESSION |
| Report |
Kovalskaya N, Hammond RW. ( 2009) Protein Expression and Purification, 1, 12-17 |
| Project Aim |
Expression and charactrization |
| Fusion |
C-terminal hexahistidine tag |
Protein Expression and
Production |
Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host |
E coli |
| Expression Strain |
BL21 (DE3) |
| Expression Temperature |
37 |
| Expression Time |
7 h |
| Expression Vector |
pET26b+sn1 |
| Expression Protocol |
Escherichia coli strain BL21 (DE3) (Stratagene, La Jolla, CA) was used as a host for expression of the target genes. The transformation of pET26b+sn1, pET26b+pth1, pET26b+sn1thrHis, and pET26b+pth1thrHis into E. coli was performed according to the manufacturer’s instructions. The induction procedure for gene expression was as follows: 2 ml of Luria–Bertani broth (LB) [20 g of Bacto tryptone; 10 g of Bacto yeast extract and 20 g of NaCl per liter of H2O] containing kanamycin (50 lg/ml) was inoculated with a bacterial colony and incubated overnight at 225 rpm at 37 °C. Five hundred microliters of overnight culture were transferred into a flask containing 50 ml of LB medium with the same antibiotic (50 lg/ml), and agitated at 225 rpm at 37 °C until the culture density reached an OD600 of 0.7–0.8. IPTG was added to final concentration 1.5 mM with subsequent incubation at 225 rpm at 37 °C for 7 h. After incubation, the bacterial cells were harvested by centrifugation in 50 ml Falcon tubes at 4000 rpm for 20 min at 4 °C and frozen at 80 °C. A non-induced culture was used as a negative control. |
| Method of Induction |
IPTG |
| Cell Density (at induction) |
OD 0.7-0.8 = 600 |
| Cell Disruption Method |
Not stated |
| Lytic Agent |
None |
| Pre-refolding Purification |
not specified |
| Solubility |
insoluble |
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REFOLDING |
| Refolding Method |
Dialysis |
| Wash Buffer |
n/a |
| Solubilization Buffer |
solubilization buffer was supplemented with 1 mM DTT |
| Refolding Buffer |
20 mM Tris–HCl pH 8.0, 0.1 mM DTT |
| Pre-refolding Purification |
not specified |
| Tag Cleaved |
no |
| Refolding pH |
8 |
| Refolding Temperature |
4 |
| Protein Concentration |
n/a |
| Refolding Time |
n/a |
| Redox Agent |
DTT |
| Redox Agent Concentration |
0.1 mM |
| Refolding Protocol |
IB purification, solubilization and refolding : IBs were purified using BugBuster Master Mix Protein Extraction Reagent (Novagen) according to a standard procedure (Novagen, User protocol TB245 Rev. E 0304, P.8). Solubilization of the subsequent IBs was carried out using the Protein Refolding Kit (Novagen). Specifically, the IBs solubilization buffer was supplemented with 1 mM DTT for correct folding of disulfide bonds and with 2% N-lauroylsarcosine to reduce hydrophobic aggregation. The solubilized proteins were dialyzed against 20 mM Tris–HCl pH 8.0 four times at 4 °C. The first and second dialysis buffers were supplemented with 0.1 mM DTT. Aliquots of the SN1 and PTH1 proteins were subjected to denaturing electrophoresis in a gradient NovexÒ Tris–Glycine Gel (10–20%; Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and visualized by staining with SimplyBlue Safe Stain (Invitrogen). The protein concentration was measured with the Quick StartTM Bradford Dye Reagent (Bio-Rad, Hercules, CA) using a standard method [33] and a microplate reader 680 (Bio-Rad). All the extracted proteins were stored at 20 °C in 50% glycerol. Protein purification with nickel–nitrilotriacetic acid (Ni–NTA) metalaffinity chromatography matrices The Ni–NTA Spin Kit (QIAGEN, Valencia, CA) was used for purification of the target proteins. The purification was carried out according to the manufacturer’s instructions. |
| Assay |
Antibacterial activity assay, Western Blot |
| Chaperones |
None |
| Additives |
None |
| Additives Concentration |
n/a |
| Refolding Yield |
n/a |
| Purity |
n/a |
| Notes |
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