Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cathepsin L |
| Abbreviated Name | Cathepsin L |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Fasciola gigantica |
| UniProt Accession | Q8T0X0 |
| 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 | 535 |
| Molecular Weight | 61159.4 |
| Pi | 5.7 |
| Molecular Weight | 61159.4 |
| Disulphides | 0 |
| Full Sequence |
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGS
SNDDL WHQWKRMYNK EYNGAVDEHR RNIWEDNVKH IQEHNLRHDV GLVTYTLGLN QLTDMTFEEF KAKYLTEMPR ASDILSHGIP YEANNRAVPD KIDWRGSGYV TTVKDQGNCG SCWAFSTTGT MEGQYMKNER TSISFSEQQL VDCSGPWGNY GCSGGLMENA YEYLKQFGLE TESSYPYTAV EGQCRYNRQL GVAKVTDYYT VHSGSEVELK NLVGAEGPAA IAVDVESDFM
MYSGGIYQSQ TCLRLNHAVL AVGYGTQGGT DYWIVKNSWG LSWGERGYIR MARNRGNMCG ISSLASLPMV ARFP
|
| Notes | GST fusion protein: full length protein without signal (pre) sequence |
| Expression | |
|---|---|
| Report | Yamasaki H, Mineki R, Murayama K, Ito A, Aoki T (2002) Int J Parasitology, 32, 1031-1042 |
| Project Aim | Structure-Function |
| Fusion | N-terminal GST + pro-sequence |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 2-3h |
| Expression Vector | pGEX-4T-1 |
| Expression Protocol | Cells were grown at 37degC, expression was induced with 1mM IPTG and incubation was over 2-3h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | phopshate buffered saline (PBS) |
| Solubilization Buffer | 50mM TrisHCl pH 8.0, 50mM NaCl, 5mM EDTA, 10mM DTT, 8M urea |
| Refolding Buffer | 50mM potassium phosphate pH 10.7, 5mM EDTA, 0.1mM GSSH, 1mM GSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 10.7 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 10microg/ml |
| Refolding Time | overnig |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.1mM |
| Refolding Protocol | Harvested cells were resuspended in chilled PBS containing 0.1% Triton X-100 and 1% sarcosine and then sonicated. The suspensions were centrifuged (15000rpm, 10min) and the pellets were rinsed several times in PBS, then suspended in 20ml of 50mM TrisHCl pH 8.0, 50mM NaCl, 5mM EDTA, 10mM DTT. Urea was added to a final concentration of 8M. The protein was then slowly added dropwise (2ml/h) into 400 volumes of refolding buffer to a final concentration of about 10microg/ml and stirred at 4degC overnight. The pH was then adjusted to pH 8.0 and the protein was concentrated |
| Refolding Assay | Enzyme activity,Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |