Refolding Record:
| Protein | |
|---|---|
| Protein Name | UvrA Protein |
| Abbreviated Name | UvrA Protein |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Escherichia coli |
| UniProt Accession | P0A698 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 955 |
| Molecular Weight | 103868.0 |
| Pi | 6.1916 |
| Molecular Weight | 103868.0 |
| Disulphides | Unknown |
| Full Sequence |
MDKIEVRGARTHNLKNINLVIPRDKLIVVTGLSGSGKSSLAFDTLYAEGQRRYVESLSAY
ARQFLSLMEKPDVDHIEGLSPAISIEQKSTSHNPRSTVGTITEIHDYLRLLFARVGEPRC
PDHDVPLAAQTVSQMVDNVLSQPEGKRLMLLAPIIKERKGEHTKTLENLASQGYIRARID
GEVCDLSDPPKLELQKKHTIEVVVDRFKVRDDLTQRLAESFETALELSGGTAVVADMDDP
KAEELLFSANFACPICGYSMRELEPRLFSFNNPAGACPTCDGLGVQQYFDPDRVIQNPEL
SLAGGAIRGWDRRNFYYFQMLKSLADHYKFDVEAPWGSLSANVHKVVLYGSGKENIEFKY
MNDRGDTSIRRHPFEGVLHNMERRYKETESSAVREELAKFISNRPCASCEGTRLRREARH
VYVENTPLPAISDMSIGHAMEFFNNLKLAGQRAKIAEKILKEIGDRLKFLVNVGLNYLTL
SRSAETLSGGEAQRIRLASQIGAGLVGVMYVLDEPSIGLHQRDNERLLGTLIHLRDLGNT
VIVVEHDEDAIRAADHVIDIGPGAGVHGGEVVAEGPLEAIMAVPESLTGQYMSGKRKIEV
PKKRVPANPEKVLKLTGARGNNLKDVTLTLPVGLFTCITGVSGSGKSTLINDTLFPIAQR
QLNGATIAEPAPYRDIQGLEHFDKVIDIDQSPIGRTPRSNPATYTGVFTPVRELFAGVPE
SRARGYTPGRFSFNVRGGRCEACQGDGVIKVEMHFLPDIYVPCDQCKGKRYNRETLEIKY
KGKTIHEVLDMTIEEAREFFDAVPALARKLQTLMDVGLTYIRLGQSATTLSGGEAQRVKL
ARELSKRGTGQTLYILDEPTTGLHFADIQQLLDVLHKLRDQGNTIVVIEHNLDVIKTADW
IVDLGPEGGSGGGEILVSGTPETVAECEASHTARFLKPML
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Claassen LA, Ahn B, Koo HS, Grossman L. (1991) J Biol Chem, 266, 11380-11387 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HB101 |
| Expression Temp | 42.0 |
| Expression Time | 2h |
| Expression Vector | pLAC1 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.1M Tris-HCl, 2M NaCl, pH 7.2 |
| Solubilization Buffer | 8M urea, 1mM EDTA, 25mM Tris-PO4, 10mM DTT, 0.2M KCl, pH 7.5 |
| Refolding Buffer | 50mM HEPES, 20% glycerol, 0.2M KCl, 0.2mM DTT, 0.1mM zinc acetate |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.03 mg/ml |
| Refolding Time | 17h |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were subjected to separation on a sucrose gradient. The isolated inclusion bodies were resuspended in solubilization buffer and subjected to gel filtration in the same buffer. The peak fractions were pooled and diluted to concentration of 30 micrograms/ml and dialysed intially for 3h at room temperature in 8M urea, 0.2M KCl, 1mM DTT, 1mM Tris-acetate, pH 7.5. Subsequently the protein was dialysed against the refolding buffer at 4 degrees C overnight. |
| Refolding Assay | DNA binding |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |