Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bryodin 1 |
| Abbreviated Name | BD1 |
| SCOP Family | Plant cytotoxins |
| Structure Notes | |
| Organism | Bryonica dioica |
| UniProt Accession | P33185 |
| 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 | 294 |
| Molecular Weight | 31788.5 |
| Pi | 8.87139 |
| Molecular Weight | 31788.5 |
| Disulphides | 0 |
| Full Sequence |
MIKLLVLWLLILTIFLKSPTVEGDVSFRLSGATTTSYGVFIKNLREALPYERKVYNIPLL
RSSISGSGRYTLLHLTNYADETISVAVDVTNVYIMGYLAGDVSYFFNEASATEAAKFVFK
DAKKKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLYYYTASSAASALLVLIQS
TAESARYKFIEQQIGKRVDKTFLPSLATISLENNWSALSKQIQIASTNNGQFESPVVLID
GNNQRVSITNASARVVTSNIALLLNRNNIAAIGEDISMTLIGFEHGLYGI
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gawlak SL, Neubauer M, Klei HE, Chang CY, Einspahr HM, Siegall CB. (1997) Biochemistry, 36, 3095-3103 |
| Project Aim | Crystallography |
| Fusion | None |
| 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 | 1.5h |
| Expression Vector | pET22b |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 1mM EDTA pH 8.0 |
| Solubilization Buffer | 100mM Tris-HCl, 5mM EDTA, 7M guanidinium chloride, pH 8.0 |
| Refolding Buffer | PBS, 0.4M L-arginine, 70mM guanidinium chloride |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.4 |
| Refolding Temperature | 25.0 |
| Protein Concentration | less than 0.1mg/ml |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The expression plasmids were transformed into BL21(DE3) and cultured until the cells reached an OD650 of 0.8-1.2 and induced with 1 mM IPTG for 90 min. Cultures were harvested by centrifugation, resuspended in TE sucrose (50 mM Tris-HCl and 1 mM EDTA, pH 8.0, 20% sucrose), and kept on ice for 15 min. Following centrifugation the pellet was osmotically shocked with ice-cold water, centrifuged, and resuspended in TE containing 0.25 mg/ml lysozyme. Tergitol (4% final volume) was added to the lysed cell solution, which was incubated at 4 C for 30 min and centrifuged. The resulting inclusion bodies were washed 3 times with TE, resuspended in 10 mL of guanidine solution (7 M guanidine hydrochloride, 100 mM Tris-HCl, pH 8.0, 5 mM EDTA), sonicated (3 ?30 s), incubated on ice (1-2 h), and then centrifuged. The denatured protein in the supernatant was rapidly refolded in phosphate-buffered saline containing 0.4 M L-arginine and 70 mM guanidine solution at a concentration of <100 mg/L. The refolded protein was dialyzed against 10 mM NaH2PO4, pH 6.5, and loaded onto a CM-Sepharose column (Pharmacia, Uppsala, Sweden). Recombinant BD1 was eluted with a 0-0.3 M NaCl gradient in 10 mM NaH2PO4, pH 6.5. The fractions were analyzed by Coomassie-stained SDS-PAGE and immunoblots stained with polyclonal anti-BD antiserum. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 25mg/L culture (fermentation), 2mg/L (shake-flask) |
| Purity | |
| Notes | |