Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cauliflour mosaic virus capsid protein |
| Abbreviated Name | CaMV |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Cauliflower Mosaic Virus |
| UniProt Accession | Q83168 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 497 |
| Molecular Weight | 56705.0 |
| Pi | 5.27 |
| Molecular Weight | 56705.0 |
| Disulphides | Unknown |
| Full Sequence |
MAESILDRTINRFWYNLGEDCLSESQFDLMIRLMEESLDGDQIIDLTSLPSDNLQVEPVM
TTTDDSISEEESEFLLAIGETSEDESDSGEEPEFEQVRMDRTGGTEIPKEEDGEPSRYNE
RKRKTTEGRYFPTQPKTIPGQKQTSMGMLNIDCQTNRRTLIDDWAAEIGLIVKTNREDYL
DPETILLLMEHKTSGIPKELIRNTRWNRTTGDIIEQVIDAMYTMFLGLNYSDNKVAEKID
EQEKAKIRMTKLQLCDICYLEEFTCDYEKNMYKTELADFPGYINQYLSKIPIIGEKALTR
FRHEANGTSIYSLGFAAKIVKEELSKICDLSKKQKKLKKFNKKCCSIGEASAEYGCKKTS
TKKYHKKRYKKKYKVYKPYKKKKKFRSGKYFKPKEKKGSKQKYCPKGKKDCRCWICNIEG
HYANECPNRQSSEKAHILQQAEKLGLQPIEEPYEGVQEVFILEYKEEEEETSTEESDDES
STSEDSDSD
|
| Notes | full lenght wildtype protein and a series of delition derivatives were refolded with the same method though different buffers |
| Expression | |
|---|---|
| Report | Chapdelaine Y and Hohn T (1998) Virus Genes, 17, 139-150 |
| Project Aim | Assembly |
| 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 | 2 h |
| Expression Vector | pET3d |
| Expression Protocol | Constructs were introduced into Escherichia coli strain BL21 (DE3) by transformation. Liquid cultures were started from several single bacterial colonies in LB medium supplemented with 0.4% glucose and containing 50 mg/ml ampicillin. The cultures were grown to A600 ? 0:6 ÿ 1:0 and expression was induced with 10 mg/ml isoproyl-û-D-thiogalactopyranosid (IPTG) for 2 h at 37 C (22). Cell suspensions were cooled on ice and spun at 5000 g for 15 min. The bacterial pellets were washed and resuspended in lysis buffer (20mM Tris-HCl, pH 7.5, 10mM EDTA, 1mM dithiothreitol (DTT) and 1mM phenylmethylsulfonyl ¯uoride (PMSF, (23)) before being treated with lysozyme (2 mg/ml of culture) for 30 min on ice, and frozen on dry ice. After thawing, lysates were sonicated and spun at 10,000 g for 10 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-1.0 = 600 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20mM Tris-HCl, pH 7.5, 10mM EDTA, 1mM DTT ,1 mM phenylmethylsulfonyl fluoride (PMSF) |
| Solubilization Buffer | 8 M urea |
| Refolding Buffer | PBS; 10 mM Na2HPO4+ KH2PO4, PH 7.3, 140 mM NaCl, 5.4 mM KCl |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.3 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | Overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | 1 mM,1 mM,1 mM,1 mM,1 mM,1 mM |
| Refolding Protocol | Inclusion body pellets were washed, resolubilized in 8M urea and renatured by dialysis overnight at 4 C against the lysis buffer (containing 0.1mM PMSF and 0.1% Tween-20) OR PBS; 10 mM Na2HPO4 + KH2Po4, 140 mM NaCl, 5.4 mM KCl. After dialysis, samples were clarified by centrifugation (10,000 g for 10 min). The supernatant contained between 0.5 to 2 mg/ml of recombinant pIV. Renatured proteins (200 mg) were loaded on the top of 10±60% sucrose gradients and centrifuged at 40,000 rpm in a SW40 rotor for 4 h at 20 C. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Applied to construct pIV77-332 only. |