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| Refolding Record [Created: 2008-11-24 Updated: 2008-11-24 ] |
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PROTEIN |
| Protein Name |
Hen egg- white lysozyme |
| Abbreviated Name |
HEWL |
| SCOP Family |
C-type Lysozyme |
| Structure Notes |
n/a |
| Organism |
Chicken (Gallus gallus) |
| UniProt Accession |
P00698 |
| SCOP Unique ID |
53962 |
| Structure Solved |
y |
| Class |
Alpha+Beta |
| Molecularity |
Monomer |
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CONSTRUCT |
| Full Length |
y |
| Domain |
n/a |
| Chimera |
n/a |
| Variants |
n/a |
| Chain Length |
129 |
| Molecular Weight |
14313.1 |
| pI |
9.32 |
| Disulphides |
4 |
Pfam Domain(s) and
Number of Occurrences |
Pfam Entry
Lys(1) |
| Domain Graphics |
 |
| Full Sequence |
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVS
DGNGMNAWVAWRNRCKGTDVQAWIRGCRL
BLAST against Refold database | BLAST against NCBI NR database
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| Notes |
n/a |
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EXPRESSION |
| Report |
Nara et al., ( 2008) Colloids Surf B Biointerfaces., 1, 1 |
| Project Aim |
Protein refolding |
| Fusion |
None |
Protein Expression and
Production |
Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host |
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| Expression Strain |
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| Expression Temperature |
0 |
| Expression Time |
0 |
| Expression Vector |
|
| Expression Protocol |
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| Method of Induction |
Not Stated |
| Cell Density (at induction) |
n/a |
| Cell Disruption Method |
None |
| Lytic Agent |
None |
| Pre-refolding Purification |
not specified |
| Solubility |
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REFOLDING |
| Refolding Method |
zeolite assisted refolding |
| Wash Buffer |
n/a |
| Solubilization Buffer |
20 mM Tris–HCl [pH 7.5], 500 mM NaCl, and 6 M Gdn-HCl) containing 50 mM DTT |
| Refolding Buffer |
50 mM Tris–HCl [pH 8.5], 500 mM NaCl, 5 mg/mL PEG 20000, 1 mM EDTA, 10 mM Cys, and 1 mM cystine) containing Gdn-HCl and/or Arg |
| Pre-refolding Purification |
not specified |
| Tag Cleaved |
no tag |
| Refolding pH |
8.5 |
| Refolding Temperature |
4 |
| Protein Concentration |
n/a |
| Refolding Time |
n/a |
| Redox Agent |
Cysteine/Cystine |
| Redox Agent Concentration |
10 mM/1 mM |
| Refolding Protocol |
Adsorption of denatured/reduced lysozyme on zeolite: An aliquot of zeolite (20 mg) was equilibrated with 500 μL of denaturing buffer for 10 min. Then, 500 μL of 4 mg/mL denatured/reduced lysozyme was added and incubated for varying amounts of time at room temperature on a Rotator RT-50 (Taitec Corporation, Saitama, Japan). The zeolite was removed by centrifugation and the protein concentration of the supernatant was measured. The amount of protein adsorbed was calculated by subtracting the amount of protein in the supernatant after centrifugation from the amount of lysozyme before adsorption. Refolding lysozyme: An aliquot of zeolite (50 mg) was equilibrated with 500 μL of denaturing buffer for 10 min. Then, 500 μL of denatured lysozyme solution (2 mg/mL) was added and incubated for 1 h at room temperature. The lysozyme-adsorbed zeolite samples were washed four times with 5 mM Tris–HCl (pH 7.5). The elution and refolding of lysozyme were initiated by incubating it with refolding buffer (50 mM Tris–HCl [pH 8.5], 500 mM NaCl, 5 mg/mL PEG 20000, 1 mM EDTA, 10 mM Cys, and 1 mM cystine) containing Gdn-HCl and/or Arg at 4 °C. The zeolite was removed by centrifugation and the lysozyme activity and concentration in the supernatant were measured. All experiments were repeated at least three times. |
| Assay |
Far-UV Circular Dichroism |
| Chaperones |
None |
| Additives |
None |
| Additives Concentration |
n/a |
| Refolding Yield |
n/a |
| Purity |
n/a |
| Notes |
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