Refolding Record:
| Protein | |
|---|---|
| Protein Name | Procathepsin L |
| Abbreviated Name | Cathepsin L |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P07711 |
| 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 | 304 |
| Molecular Weight | 34446.5 |
| Pi | 5.41561 |
| Molecular Weight | 34446.5 |
| Disulphides | 3 |
| Full Sequence |
T KWKAMHNRLY GMNEEGWRRA VWEKNMKMIE LHNQEYREGK HSFTMAMNAF GDMTSEEFRQ VMNGFQNRKP RKGKVFQEPL FYEAPRSVDW REKGYVTPVK NQGQCGSCWA FSATGALEGQ MFRKTGRLIS LSEQNLVDCS GPQGNEGCNG GLMDYAFQYV QDNGGLDSEE SYPYEATEES CKYNPKYSVA NDTGFVDIPK QEKALMKAVA TVGPISVAID AGHESFLFYK EGIYFEPDCS SEDMDHGVLV VGYGFESTES DNNKYWLVKN SWGEEWGMGG YVKMAKDRRN HCGIASAASY PTV
|
| Notes | Different constructs refolded: Full protein CtsLNdelta29 (except -12 aa or Pro region) Sequence: (NB.does not include 11 N-terminal a.a. of T7 gene 10 fusion plus 3aa.linker) T KWKAMHNRLY GMNEEGWRRA 50 VWEKNMKMIE LHNQEYREGK HSFTMAMNAF GDMTSEEFRQ VMNGFQNRKP 100 RKGKVFQEPL FYEAPRSVDW REKGYVTPVK NQGQCGSCWA FSATGALEGQ 150 MFRKTGRLIS LSEQNLVDCS GPQGNEGCNG GLMDYAFQYV QDNGGLDSEE 200 SYPYEATEES CKYNPKYSVA NDTGFVDIPK QEKALMKAVA TVGPISVAID 250 AGHESFLFYK EGIYFEPDCS SEDMDHGVLV VGYGFESTES DNNKYWLVKN 300 SWGEEWGMGG YVKMAKDRRN HCGIASAASY PTV Number of amino acids: 304 Molecular weight: 34446.5 Theoretical pI: 5.42 CtsLNdelta87: -80aa. from pro-region (excluding 11 N-terminal a.a. of T7 gene 10 fusion plus 3aa.linker) RKPRKGKVFQEPL FYEAPRSVDW REKGYVTPVK NQGQCGSCWA FSATGALEGQ MFRKTGRLIS LSEQNLVDCS GPQGNEGCNG GLMDYAFQYV QDNGGLDSEE 200 SYPYEATEES CKYNPKYSVA NDTGFVDIPK QEKALMKAVA TVGPISVAID 250 AGHESFLFYK EGIYFEPDCS SEDMDHGVLV VGYGFESTES DNNKYWLVKN 300 SWGEEWGMGG YVKMAKDRRN HCGIASAASY PTV Number of amino acids: 236 Molecular weight: 26174.1 Theoretical pI: 4.95 CtsLNdelta115: No pro-sequence, -2aa. from mature protein (excluding 11 N-terminal a.a. of T7 gene 10 fusion plus 2aa.linker) RSVDW REKGYVTPVK NQGQCGSCWA FSATGALEGQ 150 MFRKTGRLIS LSEQNLVDCS GPQGNEGCNG GLMDYAFQYV QDNGGLDSEE 200 SYPYEATEES CKYNPKYSVA NDTGFVDIPK QEKALMKAVA TVGPISVAID 250 AGHESFLFYK EGIYFEPDCS SEDMDHGVLV VGYGFESTES DNNKYWLVKN 300 SWGEEWGMGG YVKMAKDRRN HCGIASAASY PTV Number of amino acids: 218 Molecular weight: 24001.5 Theoretical pI: 4.68 CtsLCdelta22: -12 aa from pro-sequence, -22aa from C-terminus, with an extra lysine at the end (excluding 11 N-terminal aa of T7 gene 10 fusion plus 3aa.linker) T KWKAMHNRLY GMNEEGWRRA 50 VWEKNMKMIE LHNQEYREGK HSFTMAMNAF GDMTSEEFRQ VMNGFQNRKP 100 RKGKVFQEPL FYEAPRSVDW REKGYVTPVK NQGQCGSCWA FSATGALEGQ 150 MFRKTGRLIS LSEQNLVDCS GPQGNEGCNG GLMDYAFQYV QDNGGLDSEE 200 SYPYEATEES CKYNPKYSVA NDTGFVDIPK QEKALMKAVA TVGPISVAID 250 AGHESFLFYK EGIYFEPDCS SEDMDHGVLV VGYGFESTES DNNKYWLVKN 300 SWGEEWGMGG YK Number of amino acids: 283 Molecular weight: 32216.9 Theoretical pI: 5.15 CtsLCC>A: -12aa of pro-sequence, C332A mutation (excluding 11 N-terminal aa of T7 gene 10 fusion plus 3aa.linker) T KWKAMHNRLY GMNEEGWRRA 50 VWEKNMKMIE LHNQEYREGK HSFTMAMNAF GDMTSEEFRQ VMNGFQNRKP 100 RKGKVFQEPL FYEAPRSVDW REKGYVTPVK NQGQCGSCWA FSATGALEGQ 150 MFRKTGRLIS LSEQNLVDCS GPQGNEGCNG GLMDYAFQYV QDNGGLDSEE 200 SYPYEATEES CKYNPKYSVA NDTGFVDIPK QEKALMKAVA TVGPISVAID 250 AGHESFLFYK EGIYFEPDCS SEDMDHGVLV VGYGFESTES DNNKYWLVKN 300 SWGEEWGMGG YVKMAKDRRN HAGIASAASY PTV Number of amino acids: 304 Molecular weight: 34414.4 Theoretical pI: 5.42 |
| Expression | |
|---|---|
| Report | Smith SM, Gottesman MM (1988) J Biol Chem, 264, 20487-20495 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3-5h |
| Expression Vector | pAR2113 |
| Expression Protocol | Cells were grown in LB medium containing 0.05-0.1mg/ml ampicillin. Broth was inoculated with a 1:25 dilution of overnight culture and grown to mid-log phase, when protein expression was induced by the addition 0.4mM IPTG, cultures were incubated for a further 3-5h. Cells were harvested by centifugation and stored at -70degC. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Osmotic shock + sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 10mM TrisHCl pH 8.0, 1mM EDTA |
| Solubilization Buffer | 50mM TrisHCl pH 8.0, 8M urea, 50mM NaCl, 5mM EDTA, 10mM DTT |
| Refolding Buffer | 50mM potassium phosphate pH 10.7, 5mM EDTA, 1mM reduced glutathione, 0.1mM oxidized glutathione |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 10.7 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 12-16h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1mM/0.1mM,1mM/0.1mM,1mM/0.1mM |
| Refolding Protocol | The cells were thawed and subjected to osmotic shock by 15min incubations at 4degC firstly in sucrose buffer (5mM TrisHCl, pH 7.6, 20% sucrose, 1mM EDTA), and then in 1mmM EDTA, with centrifugation (15min x 8700g) following each incubation. The pelleted spheroplasts were then resuspended in cold wash buffer and disrupted by sonication (3x30s). The mixture was centrifuged (20min, 17000g) and the pelleted inclusion bodies were then washed twice more in wash buffer. The inclusion bodies were resuspended in solubilization buffer (3ml buffer/g bacterial cells) and incubated at 56degC for 2h, followed by two rounds of centrifugation (2x 20min, 17000g). The supernatant was then slowly added dropwise (2ml/h) into 200 volumes of refolding buffer and stirred overnight at 4degC. The refolded mixture was then adjusted to pH 8.0 using HCl and concentrated in an ultrafiltration unit. Precipitated protein was then removed by centrifugation (20min x 17000g), and the soluble refolded protein was then loaded onto a Sephadex G-75 column equilibrated with 10mM potassium phosphate pH 8.0. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | 0.5mg/4L |
| Purity | |
| Notes | |