|
|
|
|
|
|
|
|
|
| Refolding Record [Created: 2008-12-01 Updated: 2008-12-01 ] |
|
|
|
|
PROTEIN |
| Protein Name |
Cysteine protease falcipain-2 |
| Abbreviated Name |
n/a |
| SCOP Family |
Papain-like Cysteine Proteinases |
| Structure Notes |
n/a |
| Organism |
Plasmodium falciparum |
| UniProt Accession |
Q9NBD4 |
| SCOP Unique ID |
142847 |
| Structure Solved |
y |
| Class |
Alpha+Beta |
| Molecularity |
Unknown |
|
CONSTRUCT |
| Full Length |
y |
| Domain |
n/a |
| Chimera |
n/a |
| Variants |
n/a |
| Chain Length |
484 |
| Molecular Weight |
55992.3 |
| pI |
6.83 |
| Disulphides |
Unknown |
Pfam Domain(s) and
Number of Occurrences |
Pfam Entry
Inhibitor_I29(1), Peptidase_C1(1) |
| Domain Graphics |
 |
| Full Sequence |
MDYNMDYAPHEVISHQGERFVDKYVDRKILKNKKSLLVIISLSVLSVVGFILFYFTPNFRKSDLFKNSSVENNNDDYIINSLLKSPNGKKFIVSKIDEAL
SFYDSKKNDINKYNEGNNNNNADFKGLSLFKENTPSNNFIHNKDYFINFFDNKFLMNNAEHINQFYMFIKTNNKQYNSPNEMKERFQVFLQNAHKVNMHN
NNKNSLYKKELNRFADLTYHEFKNKYLSLRSSKPLKNSKYLLDQMNYEEVIKKYRGEENFDHAAYDWRLHSGVTPVKDQKNCGSCWAFSSIGSVESQYAI
RKNKLITLSEQELVDCSFKNYGCNGGLINNAFEDMIELGGICPDGDYPYVSDAPNLCNIDRCTEKYGIKNYLSVPDNKLKEALRFLGPISISVAVSDDFA
FYKEGIFDGECGDELNHAVMLVGFGMKEIVNPLTKKGEKHYYYIIKNSWGQQWGERGFINIETDESGLMRKCGLGTDAFIPLIE
BLAST against Refold database | BLAST against NCBI NR database
|
| Notes |
n/a |
|
EXPRESSION |
| Report |
Sijwali et al., ( 2001) Protein Expression and Purification, 22, 128-134 |
| Project Aim |
Protein refolding |
| Fusion |
None |
Protein Expression and
Production |
Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host |
E coli |
| Expression Strain |
BL21 (DE3) |
| Expression Temperature |
37 |
| Expression Time |
5 h |
| Expression Vector |
pQ-35FP2 |
| Expression Protocol |
After optimization of small-scale expression, large scale expression of falcipain-2 was carried out as follows. Bacteria containing pQ-35FP2 were grown to midlog phase at 37C, IPTG (0.25 mM) was added, and growth was continued for 5 h at 37C. Cells (23 g wet wt) were then washed with ice-cold 100 mM Tris –Cl, 10 mM EDTA, pH 7.4 (5 ml/g wet wt of the cells), soni
cated (12 cycles of 10 s each, with cooling for 10 s between the cycles), and centrifuged at 12,000g for 30 min at 4C. The pellet was washed twice with 2.0 M urea, 20 mM Tris –Cl, 2.5% Triton X-100, pH 8.0 (5 ml/g wet wt of the cells), washed twice with 20% sucrose, 20 mM Tris –Cl, pH 8.0 (5 ml/g wet wt of the cells), centrifuged out a systematic optimization of the expression and at 17,000g for 30 min at 4C, and solubilized in 6 M guanidine –HCl, 20 mM Tris –Cl, 500 mM NaCl, 10 mM expression and imidazole, pH 8.0 (5 ml/g of inclusion body pellet) at room temperature for 60 min with gentle stirring. |
| Method of Induction |
IPTG |
| Cell Density (at induction) |
n/a |
| Cell Disruption Method |
Sonication |
| Lytic Agent |
None |
| Pre-refolding Purification |
Ni-NTA agrose chromatography |
| Solubility |
insoluble |
|
REFOLDING |
| Refolding Method |
Dilution |
| Wash Buffer |
2.0 M urea, 20 mM Tris –Cl, 2.5% Triton X-100, pH 8.0 |
| Solubilization Buffer |
6 M guanidine –HCl, 20 mM Tris –Cl, 500 mM NaCl, 10 mM expression and imidazole, pH 8.0 |
| Refolding Buffer |
100 mM Tris –Cl, 1 mM EDTA, 30% glycerol, 250 mM L-arginine, 1 mM GSH, 1 mM GSSG, pH 9.0 |
| Pre-refolding Purification |
Ni-NTA agrose chromatography |
| Tag Cleaved |
no tag |
| Refolding pH |
9 |
| Refolding Temperature |
4 |
| Protein Concentration |
n/a |
| Refolding Time |
n/a |
| Redox Agent |
GSH/GSSG |
| Redox Agent Concentration |
1/1 mM |
| Refolding Protocol |
Ni –NTA-purified falcipain-2 was reduced with 10 mM dithiothreitol (DTT) at 37C for 45 min. Refolding of recombinant falcipain-2 to an active protease was then assessed using 203 refolding buffers differing in the concentration of redox couple (reduced glutathione (GSH) to oxidized glutathione (GSSG)), aggregation suppressors (L-arginine, KCl, nondetergent sulfobetaine (NDSB-256) (CalBiochem), Triton X-100, and polyethylene glycol), and cosolvents (glycerol and sucrose), and a rapid dilution method (18) was adapted to a microtiter format. Refolding was initiated by 100-
fold dilution of ice-cold reduced–denatured protein to a final protein concentration of 10 ug/ml in 350 ul of ice-cold refolding buffer followed by incubation at 4C for 20 h. Refolding efficiency was evaluated by assaying 20 ul of each refolding reaction for hydrolysis of benzyloxycarbonyl-Phe-Arg-7-amino-4-methyl coumarin (Z- Phe-Arg-AMC) as described below, and the efficiency for each reaction was defined as the percentage of the maximum rate of hydrolysis achieved in this experiment. Refolding efficiency of the best refolding buffer was further assessed at different pHs and varied concentrations of glycerol and L-arginine. |
| Assay |
Activity assay |
| Chaperones |
None |
| Additives |
L-Arginine, Glycerol |
| Additives Concentration |
30%/250mM |
| Refolding Yield |
n/a |
| Purity |
n/a |
| Notes |
|
|
|
|
