Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysozyme |
| Abbreviated Name | Lysozyme |
| SCOP Family | C-type Lysozyme |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P61626 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 130 |
| Molecular Weight | 14700.7 |
| Pi | 9.27 |
| Molecular Weight | 14700.7 |
| Disulphides | 4 |
| Full Sequence |
KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Li M, Su Z (2002) Chromatographia, 56, 33-38 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 28.0 |
| Expression Time | 7 h |
| Expression Vector | pBV-HLy |
| Expression Protocol | E. Coli BL21 was the host cell for plasmid pBV-Hly (a gift plasmid from Prof. N.J. Qian, Institute of Microbiology, Chinese Academy of Sciences), which was cultured in RM culture medium at 28 c for 4 h [20]. Product formation was induced by temperature shift from 28 to 42 c and in- |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 0.1 mol U 1 Tris-HC1 at pH 8.0 containing 0.5 mmol L 1 EDTA and 0.5% (v/v) Triton X-100 |
| Solubilization Buffer | 8 moll 1 urea, 0.05moll 1 Tris-HC1 (pH 8.0), and 0.1 mol L 1 dithiothreitol (DTT). |
| Refolding Buffer | buffer A (0.05 mol/ L Tris-HC1 at pH 8.0 containing 2 mol/ L urea, 3 mmol/ L GSH and 0.3 mmol/ L GSSG)buffer B (0.1 mol/ L Tris-HC1 at pH 8.0 containing 0.2 mol/ L (NH4)2804, 2 mol/ Lurea, 3 mmol/ L GSH and 0.3 mmol/ L GSSG) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 3/0.3 mM |
| Refolding Protocol | Cation exchange refolding without any gradient was conducted on a 7 mL column packed with SP Sepharose Fast Flow (Pharmacia Biotech, Sweden). After pre-equilibrated with buffer A (0.05 mol L 1 Tris-HC1 at pH 8.0 containing 2 mol/ L urea, 3 mmol/ L GSH and 0.3 mmol/ L GSSG), the column was loaded with the required volume of protein solution. After rinsing with buffer A for two-column volumes, the column was eluted with buffer B (0.1 mol/ L Tris-HC1 at pH 8.0 containing 0.2 mol/ L (NH4)2804, 2 mol/ Lurea, 3 mmol/ L GSH and 0.3 mmol/ LGSSG). All the operations were carried out on an AKTA Purifier Workstation (Pharmacia). The flow rate was controlled at 0.4 mL min 1. For chromatographic refolding with a single gradient of urea concentration, the same column was used but the concentration of urea in buffer A was 6 mol L 1 and 1 mol L 1 urea in buffer B. After DTT in the loaded protein sample was rinsed with buffer A, the bound protein was eluted by gradient increase of buffer B concentration from 0 to 100% (v/v, Buffer B/Buffer A) within the pre-set length of the elution process. |
| Refolding Assay | Protein activity assay |
| Refolding Chaperones | None |
| Refolding Additives | (NH4)2SO4 |
| Additives Concentration | 0.2 |
| Refolding Yield | 87% |
| Purity | n/a |
| Notes | n/a |