Refolding Record:
| Protein | |
|---|---|
| Protein Name | HIV-1 Rev binding protein-like |
| Abbreviated Name | HIV-1 REV |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | A4D2D6 |
| 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 | 481 |
| Molecular Weight | 48963.1 |
| Pi | 9.28 |
| Molecular Weight | 48963.1 |
| Disulphides | Unknown |
| Full Sequence |
MVMAAKKGPGPGGGVSGGKAEAEAASEVWCRRVRELGGCSQAGNRHCFECAQRGVTYVDITVGSFVCTTCSGLLRGLNPPHRVKSISMTTFTEPEVVFLQSRGNEVCRKIWLGLFDARTSLVPDSRDPQKVKEFLQEKYEKKRWYVPPDQVKGPTYTKGSASTPVQGSIPEGKPLRTLLGDPAPSLSVAASTSSQPVSQSHARTSQARSTQPPPHSSVKKASTDLLADIGGDPFAAPQMAPAFAAFPAFGGQTPSQGGFANFDAFSSGPSSSVFGSLPPAGQASFQAQPTPAGSSQGTPFGATPLAPASQPNSLADVGSFLGPGVPAAGVPSSLFGMAGQVPPLQSVTMGGGGGSSTGLAFGAFTNPFTAPAAQSPLPSTNPFQPNGLAPGPGFGMSSAGPGFPQAVPPTGAFASSFPAPLFPPQTPLVQQQNGSSFGDLGSAKLGQRPLSQPAGISTNPFMTGPSSSPFASKPPTTNPFL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dangerfield JA, Hohenadl C, Egerbacher M, Kodajova P, Salmons B, Günzburg WH. (2005) Gene, 358, 17-31 |
| Project Aim | Over expression & Renaturation |
| 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 | 3 h |
| Expression Vector | pET3a |
| Expression Protocol | Production of recombinant Rev protein Using PCR, the rev gene from was inserted via the multiple cloning site into the bacterial expression vector pET–3a (Stratagene) to create pETsRev. Using a modified version of a PCR mutagenesis method (Vallette et al., 1989), six histidine residues were fused in frame immediately downstream of the rev gene (C-terminal His tag) to create pETsRevCHis. pETsRevCHis was transformed into BL21(DE3) bacteria (Stratagene) and inclusion bodies were purified from bacterial lysates after 3 h of stimulation at 37 °C with 1 mM IPTG. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: hydrophobic interaction chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidine hydrochloride (Gdn–HCl) |
| Refolding Buffer | 6 M to 0 M urea |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion bodies were disrupted using 6 M guanidine hydrochloride and the denatured protein solution was loaded onto HiTrap Chelating HP sepharose columns (Amersham) and subjected to a 6 M to 0 M urea refolding flow gradient controlled by an Äktaprime protein purification unit (Amersham). Eluted fractions were tested by Western blot using a rat anti-Rev antibody (a kind gift from Andrea Kleinschmidt, GSF, Neuherberg) to ascertain the quantity and integrity of Rev protein. Suitable fractions were ultra-filtered (VIVASPIN 2, Sartorius) to concentrate. An aliquot of the final purified and concentrated Rev protein was tested by Western blot and Coomassie gel and shown to be of > 99% purity. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 99% |
| Purity | n/a |
| Notes | n/a |