Refolding Record:
| Protein | |
|---|---|
| Protein Name | p58 natural killer cell inhibitory receptor |
| Abbreviated Name | p58 NK receptor |
| SCOP Family | I set domains |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P43626 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 200 |
| Molecular Weight | 22041.7 |
| Pi | 6.03394 |
| Molecular Weight | 22041.7 |
| Disulphides | 2 |
| Full Sequence |
HEGVHRKPS LLAHPGPLVK SEETVILQCW SDVMFEHFLL HREGMFNDTL RLIGEHHDGV SKANFSISRM TQDLAGTYRC
YGSVTHSPYQ VSAPSDPLDI VIIGLYEKPS LSAQPGPTVL AGENVTLSCS SRSSYDMYHL SREGEAHERR LPAGPKVNGT FQADFPLGPA THGGTYRCFG SFHDSPYEWS KSSDPLLVSV T
|
| Notes | Two extracellular domain constructs expressed and refolded: 2) sol-cl42H: a.a.1-224 HEGVHRKPS LLAHPGPLVK SEETVILQCW 50 SDVMFEHFLL HREGMFNDTL RLIGEHHDGV SKANFSISRM TQDLAGTYRC 100 YGSVTHSPYQ VSAPSDPLDI VIIGLYEKPS LSAQPGPTVL AGENVTLSCS 150 SRSSYDMYHL SREGEAHERR LPAGPKVNGT FQADFPLGPA THGGTYRCFG 200 SFHDSPYEWS KSSDPLLVSV TGNPSNSWPS PTEPSSKTGN PRHLH Number of amino acids: 224 Molecular weight: 24608.4 Theoretical pI: 6.26 |
| Expression | |
|---|---|
| Report | Fan QR, Garboczi DN, Winter CC, Wagtmann N, Long EO, Wiley DC (1996) Proc. Natl. Acad. Sci. USA, 93, 7178-7183 |
| Project Aim | Functional Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pLM-1 |
| Expression Protocol | Cells were grown at 37degC in LB medium containing 100microg/ml ampicilin, 34microg/ml chloramphenicol, 0.4% glucose. When cells reached logarithmic phase, protein expression was induced with 0.5mM IPTG. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | not stated |
| Solubilization Buffer | 8M urea, 25mM Mes pH 6.0, 10mM EDTA, 1mM DTT |
| Refolding Buffer | 100mM TrisHCl pH 8.3, 500mM L-arginineHCl, 2mM EDTA, 6.4mM cysteamine, 3.6mM cystamine, 0.1mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.3 |
| Refolding Temperature | 10.0 |
| Protein Concentration | 6microM |
| Refolding Time | 72-90h |
| Redox Agent | cysteamine/cystamine |
| Redox Agent Concentration | 6.4mM/3.6mM,6.4mM/3.6mM |
| Refolding Protocol | Cells were harvested by centrifugation, resuspended in 50mM TrisHCl pH 8.0, 25% sucrose, 1mM EDTA, and lysed by freezing and thawing in the presence of 1mg/ml lysozyme. Inclusion bodies were collected and washed extensively, then dissolved in solubilization buffer. The solubilized protein was diluted into 1L of refolding buffer. The final concentration of protein was 6microM. The refolding mixture was incubated at 10degC for 72-90h. The mixture then was dialyzed twice, first against 10L of 100mM urea at 4degC for 24h and then against 10L of 10mM TrisHCl, pH7.5, 10mM Mes 100mM urea at 4degC for 24h. The protein was then purified by anion exchange chromatography and gel filtration. |
| Refolding Assay | Gel filtration chromatography |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine |
| Additives Concentration | 0.5M |
| Refolding Yield | 20-37% |
| Purity | |
| Notes | |