Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bovine serum albumin |
| Abbreviated Name | BSA |
| SCOP Family | Serum albumin-like |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P02769 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 583 |
| Molecular Weight | 66433.0 |
| Pi | 5.59869 |
| Molecular Weight | 66433.0 |
| Disulphides | >10 |
| Full Sequence |
DTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Langenhof M, Leong SSJ, Pattenden LK, Middelberg APJ (2005) Journal of Chromatography A, 1069, 195-201 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | column refolding: ion-exchange chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 50mM TrisHCl, 3mM EDTA, 8M urea, 0.1M DTT, pH 8.5 |
| Refolding Buffer | 50mM TrisHCl, 1mM EDTA, 79mM urea, 1.1mM GSSG, 2.2mM GSH pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.5-20mg/ml |
| Refolding Time | 40h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2.2/1.1mM,2.2/1.1mM |
| Refolding Protocol | The protein was denatured and reduced by incubating a 20-100mg/ml solution for at least 3h at room temperature in denaturation buffer. A HiTrap Q-sepharose column was equilibrated with denaturation buffer without DTT (buffer A), then 1ml unfolded protein at 0.5-20mg/ml in denaturation buffer was loaded onto the column. The column was washed with 5 volumes of buffer A, then the protein was refolded by washing with refolding buffer. The flow rate was then stopped, and the protein was left on the column to incubated for up to 40h, followed by washing with 6 column volumes of Tris-EDTA buffer (50mM TrisHCl, 1mM EDTA, pH 8.5). The protein was then eluted with a 20 column volume linea gradient in Tris-EDTA buffer from 0-1M NaCl. |
| Refolding Assay | Far-UV Circular Dichroism,HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 67% |
| Purity | n/a |
| Notes | lower yield compared to dilution (record 7m4r5) but can refold at higher protein concentrations |