Refolding Record:
| Protein | |
|---|---|
| Protein Name | Leukemia inhibitory factor receptor |
| Abbreviated Name | LIF |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P42702 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Extracellular domain aa.1-833 |
| Chimera | LIF signal sequence and extracellular domain fused to Decay accelerating factor last 37 aa |
| Variants | n/a |
| Chain Length | 876 |
| Molecular Weight | 99269.9 |
| Pi | 7.16358 |
| Molecular Weight | 99269.9 |
| Disulphides | 1 |
| Full Sequence |
HHHHHH MMDIYVCLKR PSWMVDNKRM RTASNFQWLL STFILLYLMN QVNSQKKGAP HDLKCVTNNL QVWNCSWKAP SGTGRGTDYE VCIENRSRSC YQLEKTSIKI PALSHGDYEI TINSLHDFGS STSKFTLNEQ NVSLIPDTPE ILNLSADFST STLYLKWNDR GSVFPHRSNV IWEIKVLRKE SMELVKLVTH NTTLNGKDTL HHWSWASDMP LECAIHFVEI RCYIDNLHFS GLEEWSDWSP VKNISWIPDS QTKVFPQDKV ILVGSDITFC CVSQEKVLSA LIGHTNCPLI HLDGENVAIK IRNISVSASS GTNVVFTTED NIFGTVIFAG YPPDTPQQLN CETHDLKEII CSWNPGRVTA LVGPRATSYT LVESFSGKYV RLKRAEAPTN ESYQLLFQML PNQEIYNFTL NAHNPLGRSQ STILVNITEK VYPHTPTSFK VKDINSTAVK LSWHLPGNFA KINFLCEIEI KKSNSVQEQR NVTIKGVENS SYLVALDKLN PYTLYTFRIR CSTETFWKWS KWSNKKQHLT TEASPSKGPD TWREWSSDGK NLIIYWKPLP INEANGKILS YNVSCSSDEE TQSLSEIPDP QHKAEIRLDK NDYIISVVAK NSVGSSPPSK IASMEIPNDD LKIEQVVGMG KGILLTWHYD PNMTCDYVIK WCNSSRSEPC LMDWRKVPSN STETVIESDE FRPGIRYNFF LYGCRNQGYQ LLRSMIGYIE ELAPIVAPNF TVEDTSADSI LVKWEDIPVE ELRGFLRGYL FYFGKGERDT SKMRVLESGR SDIKVKNITD ISQKTLRIAD LQGKTSYHLV LRAYTDGGVG PEKSMYVVTK ENS PNKGSGT TSGTTRLLSG HTCFTLTGLL GTLVTMGLLT
|
| Notes | GST fusion protein between hexaHis tag and protein, but not included in sequence |
| Expression | |
|---|---|
| Report | Liu H, Moreau J-F, Gualde N, Fu J (1997) Mol Cell Biochem, 169, 43-50 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine and GST tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | XL1 |
| Expression Temp | 30.0 |
| Expression Time | 3-4h |
| Expression Vector | pGEX-5T |
| Expression Protocol | diluted 1-fold with fresh medium with ampicillin and incubated until A600 reached 0.5. Expression was induced with 0.5mM IPTG and further incubation for another 3-4h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.5 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Column Refolding Combination |
| Wash Buffer | 0.1M glycine-NaOH pH 9.0, 5M urea |
| Solubilization Buffer | 0.1M glycine-NaOH pH 9.0, 8M urea |
| Refolding Buffer | 50mM TrisHCl, 1mM DL-DTT, 0.5M NaCl, 0.5% Triton X-100, 1mM PMSF |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 24h |
| Redox Agent | DL-DTT |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | Cells were grown overnight at 30degC in LB medium containing 150microg/ml ampicillin, The cells were centrifuged and resuspended in 2ml 50mM TrisHCl 1mM EDTA, 100mM NaCl pH 8.0 with 1.0mg/ml lysozyme and 1mM PMSF for 20min at 0degC, followed by treatment with 1% Triton X-100 for 10min at 0degC. After sonication (2x20sec) the cell lysate was centrifuged (15000rpm, 5min, 4degC). The pellet was resuspended 2ml wash buffer and then centrifuged - this wash step was repeated twice. The pellet was then disolved in 2ml solublization buffer for 10min at room temperature and then sonicated (2x20sec). After centrifugation (15000rpm, 10min, 4degC0, the supernatant was stored at 4degC. The dissolved protein was passed down a Sephadex G-25 column equilibrated in refolding buffer. Selected fractions were collected anddiluted between 2 and 100-fold with refolding buffer, then left for a further 24h at 4degC. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |