Refolding Record:
| Protein | |
|---|---|
| Protein Name | Photosystem Q(B) protein |
| Abbreviated Name | psbA |
| SCOP Family | Photosystems [58157] |
| Structure Notes | |
| Organism | Barley (Hordeum vulgare) |
| UniProt Accession | P05337 |
| 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 | 344 |
| Molecular Weight | 37922.5 |
| Pi | 5.29 |
| Molecular Weight | 37922.5 |
| Disulphides | Unknown |
| Full Sequence |
TAILERRESTSLWGRFCNWITSTENRLYIGWFGVLMIPTLLTATSVFIIAFIAAPPVDIDGIREPVSGSLLYGNNIISGAIIPTSAAIGLHFYPIWEAASVDEWLYNGGPYELIVLHFLLGVACYMGREWELSFRLGMRPWIAVAYSAPVAAATAVFLIYPIGQGSFSDGMPLGISGTFNFMIVFQAEHNILMHPFHMLGVAGVFGGSLFSAMHGSLVTSSLIRETTENESANEGYKFGQEEETYNIVAAHGYFGRLIFQYASFNNSRSLHFFLAAWPVVGIWFTALGISTMAFNLNGFNFNQSVVDSQGRVINTWADIINRANLGMEVMHERNAHNFPLDLA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Efimov VA, Fradkov AF, Raskind AB, Khristin MS, Klimov VV, Chakhmakhcheva OG. (1994) FEBS Letters, 348, 153-7 |
| Project Aim | Recombinant Protein Expression |
| 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 | 5-7 h |
| Expression Vector | pGT7A |
| Expression Protocol | The vectors pGT7A and pVTr2A were constructed as described earlier [5,6]. Plasmid pGT7H was obtained from pGT7A as shown in Fig.1. Oligonucleotides were synthesized by the H-phosphonate method [7].E. coli strain BL2l(DE3) was used as host in theexpression experiments 181. Bacterial cultures containing expression plasmids were grown in LB medium containing ampicillin. After the addition of IPTG (1 mM) and incubation at 37°C for 5-7 h, the cells were collected and sonicated as described. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.5% Triton X-100, 20 mM Tris-HCl (pH 7.5) 1 mM ME(B-mercaptoethanol) |
| Solubilization Buffer | 8 M urea, 0.1 M Tris-HCl (pH 8.0), 2% ME |
| Refolding Buffer | 50 mM Tris-HCl, 0.1% Triton X-100 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet was solubilized in 8 M urea, 0.1 M Tris-HCl (pH 8.0), 2% ME. The recombinant Dl protein was renaturated by dialysis against 50 mM Tris-HCl, 0.1% Triton X-100, and then was applied to a Red-Sepharose column (Pharmacia). The column was washed with 50 mM Tris-HCl @H 6.8), 0.1% OGP, and the purified Dl protein was eluted with 0.3 M NaCl. In the case of Dl-His protein, the pellet obtained after sonication of cells was suspended in 6 M guanidine-HCl (PH 8.0) and loaded onto a Ni2+- NTA-agarose column (Qiagen). The purification was performed using a protocol recommended by producer. The bound Dl-His protein was renaturated using a linear gradient of 8 M urea, 0.05 M Tris-HCl (PH S.Op.05 M Tris-HCl (PH 6.8) 0.1% OGP, and then was eluted from the column by imidazole. In a parallel experiment, 0.1 mM [14C]atrazine in 0.05 M Tris-HCl, 0.1% OGP was passed through the column with the renaturated Dl-His protein. Proteins were analysed by 12% SDS-PAGE [9] and by Western blotting [6] with rabbit antibodies against mature barley Dl protein isolated as described [lO,ll]. |
| Refolding Assay | Western Blot,SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Triton X-100 |
| Additives Concentration | 0.1% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |