Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interferon gamma |
| Abbreviated Name | IFN gamma |
| SCOP Family | Interferons/interleukin-10 (IL-10) |
| Structure Notes | |
| Organism | Cavia porcellus (Guinea pig) |
| UniProt Accession | Q8CGS0 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 166 |
| Molecular Weight | 19590.4 |
| Pi | 9.58 |
| Molecular Weight | 19590.4 |
| Disulphides | Unknown |
| Full Sequence |
MKYTSSILALQFCIILSFSSYYCQSRFTNEIRILKNYFNADNSDVGDNGTLFVGILKNCQEESERKIFQSQIVSFYFKLFEKHFTDNQTVQNSMNTIKEQIITKFFKDNSSNKVQAFKNLIQISVNDEHVQRQAIIELKKVIDDLSPNQRKRRRTQMLFQSRRASK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Jeevan A, McFarland CT, Yoshimura T, Skwor T, Cho H, Lasco T, McMurray DN. (2006) Infection and Immunity, 74, 213-224 |
| Project Aim | Expression and charactrization |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pQE-30 |
| Expression Protocol | The plasmid chosen for expression of rgpIFN-γ was a pQE-30 derivative (QIAGEN, Valencia, CA), and the protocol used was essentially the same as that used earlier in our laboratory for rgpTNF-α and rgpIL-8 expression (29, 32). The plasmid was originally engineered by Magnus Hook (TAMU-HSC IBT, Houston, TX) to include a thrombin cleavage site, THR, between the six-His tag and the multiple cloning sites. gpIFN-γ cDNA provided by one of us (T.Y.) was cloned into the pQE-30 multiple cloning site. Escherichia coli strain M15 transformed with pQE-30/gpIFN-γ was grown at 37°C for approximately 1.5 h (optical density at 600 nm [OD600] = 0.7) in Luria-Bertani medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin (Sigma). Induction of protein was done with 1 mM isopropyl-B-d-thiogalactopyranoside (IPTG; Sigma), and 5 h later, the bacterial cells were harvested by centrifugation at 4,000 × g for 20 min. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.7 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 100 mM NaH2PO4, 10 mM Tris-Cl, pH 4.5) in the presence of 8 M urea |
| Refolding Buffer | 50 mM Tris, 50 mM NaCl, pH 8.0) containing 14 μl of β-mercaptoethanol |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 2-4 h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 14 uL |
| Refolding Protocol | The cell pellet was frozen at −80°C. An 18-kDa band in the insoluble fraction was identified as a putative six-His-rgpIFN-γ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Cleared lysates (CL) were prepared by lysing the pellet under denaturing conditions by the QIAGEN protocol in the presence of 10 mM benzamidine to limit protease activity. The supernatant collected after centrifugation was mixed with a 50% Ni-nitrilotriacetic acid slurry (QIAGEN) and purified on polypropylene columns (QIAGEN). The immobilized rgpIFN-γ was washed, and the protein was eluted in elution buffer (100 mM NaH2PO4, 10 mM Tris-Cl, pH 4.5) in the presence of 8 M urea. Renaturation of the denatured protein was carried out by dropwise addition of 10 ml of eluted protein to 100 ml of dilution buffer (50 mM Tris, 50 mM NaCl, pH 8.0) containing 14 μl of β-mercaptoethanol in the absence of urea. The folded protein solution was left for 2 to 4 h at room temperature (RT) and transferred to 4°C. Protein was concentrated in Amicon Ultra-15 centrifugal filter devices (Millipore Corp., Bedford, MA) by spinning 15 ml of sample for 50 min at 2,635 × g at 4°C. The protein content was determined by the Bradford assay (Bio-Rad, Richmond, CA). The refolded protein was sequenced by the Edman protein sequencing method on a Hewlett Packard G1000A automated protein sequencer (L. Dangott, Protein Chemistry Laboratory, Texas A&M University). Aliquots of rgpIFN-γ were prepared and stored at −80°C for testing the bioactivity. The level of endotoxin in the folded recombinant protein was tested by the Limulus amebocyte lysate assay (Associates of Cape Cod, Inc., Falmouth, MA) and was found to be 0.075 endotoxin units/μg (17 pg/μg) IFN-γ and was much lower than the acceptable concentration (100 pg/μg) in commercially available recombinant cytokines. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |