Refolding Record:
| Protein | |
|---|---|
| Protein Name | CC Chemokine Lymphotactin |
| Abbreviated Name | XCL1 |
| SCOP Family | Rhodopsin Like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P46094 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 333 |
| Molecular Weight | 38507.5 |
| Pi | 8.93 |
| Molecular Weight | 38507.5 |
| Disulphides | 2 |
| Full Sequence |
MESSGNPESTTFFYYDLQSQPCENQAWVFATLATTVLYCLVFLLSLVGNSLVLWVLVKYESLESLTNIFILNLCLSDLVFACLLPVWISPYHWGWVLGDFLCKLLNMIFSISLYSSIFFLTIMTIHRYLSVVSPLSTLRVPTLRCRVLVTMAVWVASILSSILDTIFHKVLSSGCDYSELTWYLTSVYQHNLFFLLSLGIILFCYVEILRTLFRSRSKRRHRTVKLIFAIVVAYFLSWGPYNFTLFLQTLFRTQIIRSCEAKQQLEYALLICRNLAFSHCCFNPVLYVFVGVKFRTHLKHVLRQFWFCRLQAPSPASIPHSPGAFAYEGASFY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Peterson FC, Elgin ES, Nelson TJ, Zhang F, Hoeger TJ, Linhardt RJ, Volkman BF. (2004) J Biol Chem, 279, 12598-12604 |
| Project Aim | Identification and Characterization |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | SG13009[pRPEP4] |
| Expression Temp | 37.0 |
| Expression Time | 3.5 h |
| Expression Vector | pQE308HT-hLtn |
| Expression Protocol | The hLtn expression plasmid, pQE308HT-hLtn, was transformed into E. coli strain SG13009[pRPEP4] (Qiagen). Cell were grown at 37 °C in LB media containing 150 µg/ml ampicillin and 50 µg/ml kanamycin until the cell density reached A600 = 0.7–1.0. Protein expression was then induced by the addition of isopropyl--D-thiogalactopyranoside to a final concentration of 1 mM. Following induction, cells were grown for another 3.5 h, harvested and stored at -80 °C until processed further. For uniform labeling with 15N, cells were grown in M9 media containing 15N-ammonium chloride as the sole nitrogen source. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.7-1.0 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 10 mM imidizole, 0.1% (v/v) 2-mercaptoethanol, 1 mM phenylmethylsufonyl fluoride |
| Solubilization Buffer | 6 M guanidinium chloride, 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidizole, 0.1% (v/v) 2-mercaptoethanol |
| Refolding Buffer | 20 mM Tris, pH 8.0, 200 mM NaCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 22.0 |
| Protein Concentration | n/a |
| Refolding Time | 3 h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 0.1% (v/v) |
| Refolding Protocol | Recombinant hLtn was found exclusively in the insoluble fraction of the harvested cells, and isolated by the following procedure for purification and refolding. Cell paste from a 1–2-liter culture was resuspended in 10 ml of lysis buffer (50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 10 mM imidizole, 0.1% (v/v) 2-mercaptoethanol, 1 mM phenylmethylsufonyl fluoride) and lysed by two passes through a French pressure cell. Inclusion bodies were isolated by centrifugation at 10,000 x g for 15 min at 4 °C, resuspended in 10 ml of solubilization buffer (6 M guanidinium chloride, 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidizole, 0.1% (v/v) 2-mercaptoethanol), disaggregated by passage through a 16-gauge needle, and clarified by centrifugation at 10,000 x g for 10 min. This protein solution was incubated with Ni2+-nitrilotriacetic acid resin (Qiagen) in batch mode for 30 min at room temperature, packed into a 5-ml disposable column, and washed with 3 x 10-ml portions of solubilization buffer. Bound Ltn was eluted with 3 x 10-ml portions of elution buffer (6 M guanidinium chloride, 100 mM sodium acetate, pH 4.5, 300 mM NaCl, 10 mM imidizole, 0.1% (v/v) 2-mercaptoethanol). Eluted fractions were pooled and dialyzed against 3 x 4 liters of 0.3% (v/v) acetic acid to remove denaturant. Separation of the affinity tag was performed by adjusting the buffer conditions to 50 mM sodium phosphate, pH 6.5, 50 mM NaCl, and 0.1% 2-mercaptoethanol, addition of a catalytic amount of tobacco etch virus protease (1,000:1), and incubation for 3 h at 22 °C. Formation of the single hLtn disulfide was accomplished by dropwise addition of the cleavage reaction into 150 ml of oxidation buffer (20 mM Tris, pH 8.0, 200 mM NaCl), followed by dialysis of the oxidation reaction against 4 liters of oxidation buffer. Oxidized hLtn was concentrated to 20 ml by ultrafiltration in an Amicon stirred-cell using a 3-kDa molecular mass cutoff membrane. Fully refolded hLtn was separated from reduced or aggregated species by reversed-phased high performance liquid chromatography. Purity and identity of all hLtn variants were confirmed by matrix-assisted laser desorption ionization mass spectrometry. |
| Refolding Assay | Mass spectrometry |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |