Refolding Record:
| Protein | |
|---|---|
| Protein Name | Mucrofibrase 1 |
| Abbreviated Name | Mucrofibrase 1 |
| SCOP Family | Hemorrhagin |
| Structure Notes | |
| Organism | Taiwan habu (Trimeresurus mucrosquamatus) |
| UniProt Accession | Q91507 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 237 |
| Molecular Weight | 25832.5 |
| Pi | 5.8513 |
| Molecular Weight | 25832.5 |
| Disulphides | 6 |
| Full Sequence |
MRGS VIGGDE CNINEHPFLV LVYYDDYQCG GTLLNEEWVL TAAHCNGKDM EIYLGVHSKK VPNKDVQRRV PKEKFFCDSS
KTYTKWNKDI MLIRLDRPVR KSAHIAPLSL PSSPPSVGSV CRVMGWGTIT SPQETYPDVP HCANINLLDY EVCRAAYAGL PATSRTLCAG ILEGGKDSCV GDSGGPLICN GQFQGIVSWG GDPCAQPREP GVCTNVFDHL DWIKGIIAGN TDVTCPL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hung CC, Chiou SH (2000) Biochemical and Biophysical Research Com, 275, 924-930 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pQE30 |
| Expression Protocol | Cells were grown at 37degC in 1L of LB medium containing 25 mg/ml kanamycin and 100 mg/ml ampicillin. When the A600nm of the growing culture reached 0.7, IPTG was added to a final concentration of 2 mM. The culture was induced for a period of up to 4 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.7 |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris-HCl, 0.5M NaCl, 8M urea pH 8.0 |
| Refolding Buffer | 50mM Tris-HCl, 5mM cysteine-HCl |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.01-0.05mg/ml |
| Refolding Time | |
| Redox Agent | Cysteine |
| Redox Agent Concentration | n/a,n/a,5mM,5mM |
| Refolding Protocol | Whole cell pellet was solubilised in solublisation buffer prior to centrifugation at 10000g. The supernatant was retained and loaded onto Ni-NTA. Subsequent to purification on Ni-NTA the protein was refolded by dilution with refolding buffer to a final protein concentration of 0.01–0.05 mg/ml. The refolding process was allowed to proceed at room temperature. Upon refolding, autolytic cleavage of the fusion peptide occurred, resulting in a product of correct molecular weight. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |