Refolding Record:
| Protein | |
|---|---|
| Protein Name | myelin oligodendrocyte glycoprotein |
| Abbreviated Name | MOG |
| SCOP Family | V set domains (antibody variable domain-like) |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | Q61885 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 128 |
| Molecular Weight | 14591.4 |
| Pi | 5.43932 |
| Molecular Weight | 14591.4 |
| Disulphides | 1 |
| Full Sequence |
GQ FRVIGPGYPI RALVGDEAEL PCRISPGKNA TGMEVGWYRS PFSRVVHLYR NGKDQDAEQA PEYRGRTELL KETISEGKVT LRIQNVRFSD EGGYTCFFRD HSYQEEAAME LKVEDPFYWV NPGVLT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Clements CS, Reid HH, Beddoe T, Tynan FE, Perugini MA, Johns TG, Bernard CC, Rossjohn J. (2003) Proc. Natl. Acad. Sci. USA, 100, 11059-64 |
| Project Aim | Crystallography |
| 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 (pREP4) |
| Expression Temp | 37.0 |
| Expression Time | 4 hours |
| Expression Vector | pQE9 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: affinity immobilization |
| Wash Buffer | see protocol below |
| Solubilization Buffer | see protocol below |
| Refolding Buffer | see protocol below |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | ~18 hours |
| Redox Agent | Beta-mercaptoethanol/GSH-GSSG |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Expression vectors Vector used for all constructs: pQE9 (Qiagen). cDNAs encoding: 1) Mouse MOG amino acid residues 1-117 of mature protein (5’BamHI/HindIII3’ sites of pQE9); Synthesis of recombinant protein (Essentially as per Qiaexpressionist 2002; Qiagen) All plasmids are transformed in E. coli strain M15(pREP4) (Qiagen) and grown under antibiotic selection (100 ?g/ml ampicillin and 25 ?g/ml kanamycin) 1) Grow a single colony of E. Coli strain M15 (pREP4) cells carrying the plasmid at 37°C overnight with agitation (250 rpm) in 50ml of LB medium. 2) Dilute 20ml of the overnight culture 1 litre of Superbroth medium and grow at 37°C until the OD600nm reaches 0.5 to 0.7. 3) Add 1 ml of 1 M isopropyl-1-thio-?-D-galactopyranoside (IPTG; final concentration 1 mM) to the cultures to induce protein expression (remove 1 ml sample as preinduced sample for analysis- used immediately or stored at –20°C). 4) Grow induced culture at 37°C for 4 hours (remove 1 ml sample as induced sample for analysis- used immediately or stored at –20°C). 5) Harvest cultures by centrifugation at 6000 rpm for 20 min. (Determine weight of the pellet) 6) Process cell pellets immediately or freeze on dry ice and store at –80°C. Preparation of cleared lysate (Essentially as per Qiaexpressionist 2002; Qiagen) 1) Thaw cell pellets on ice and resuspend in 5 ml Lysis Buffer per gram of cells. 2) Place cell lysate on wheel and rotate slowly for 1 hour at room temperature. 3) Sonicate the lysate on ice (e.g. 3×10 sec. depends on the sonicator). Lysate should become translucent and much less viscous. (French Press works better if available). 4) Remove insoluble material (clarify the lysate) by centrifuging at 10 000 x g for 30 min at room temperature. Purification/Refolding Requires the use of a BioLogic (Bio-Rad), Akta Prime (Amersham), or similar chromatography system. 1) Apply the supernatant (usually 25 to 50ml cleared lysate from 1 litre culture) directly onto a 10 ml Ni-NTA superflow (Qiagen)-or equivalent- column previously equilibrated with 5 column volumes of GuHCl lysis buffer (Flow rate 1ml/min). 2) Wash the column with Buffer A until the baseline stabilises (Abs280nm near zero). Approx. 50ml. (Flow rate 2ml/min). 3) Wash column with 50ml of Buffer B. (Flow rate 2ml/min). 4) Wash the column with 30ml Buffer A (Flow rate 2ml/min). 5) Wash the column with 30ml Buffer C (Flow rate 2ml/min). 6) Wash the column with 30ml Buffer D (Flow rate 2ml/min). 7) Wash the column with 30ml Buffer C (Flow rate 2ml/min). 8) Repeat steps 6 & 7. 9) Wash the column with 50ml Buffer A (Flow rate 2ml/min). 10) Wash the column with a linear gradient of Buffer A (containing 14 mM 2-mercaptoethanol) vs. Buffer E over 16 hours (i.e. starting with 100% Buffer A/14 mM 2-mercaptoethanol-0% Buffer E and ending in 0% Buffer A/14mM 2-mercaptoethanol-100% Buffer E). (Flow rate 1ml/min). 11) Wash the column with a linear gradient of Buffer E vs. Buffer F over 5 hours (ending in 100% Buffer F) (Flow rate 1ml/min). 12) Elute the bound protein with Buffer G (Flow rate 1ml/min). 13) Extensively dialyse the protein against a buffer suitable for your purposes (eg PBS or TBS pH8). 14) Sterilise the protein by passage through a 0.2 ?m membrane filter and store at 4°C. We do not recommend freezing –tends to drop out of solution. You can concentrate the protein to about 4mg/ml but then it starts to fall out of solution. This material always contains some aggregate (~5-10%). This can be easily removed after elution from Ni NTA column: Gel filtration on S200 16/60 (Amerham Biosciences) or equivlent followed by monoQ column (Amersham Biosciences). Solutions: Super Broth (per litre) 32 g of tryptone 20 g of yeast extract 5 g NaCl Adjust to pH 7.0 with 10M NaOH Lysis Buffer 6 M Guanidine-HCl 0.5M NaCl (or 100 mM NaH2PO4-not much difference in resulting purity) 10 mM Tris Immidazole 5 mM pH 8.0 Buffer A 8 M urea 100 mM NaH2PO4 10 mM Tris pH 8.0 Buffer B 8 M urea 100 mM NaH2PO4 10 mM Tris pH 6.3 Buffer C 10 mM Tris pH 8.0 Buffer D 60% isopropanol 10 mM Tris pH 8.0 Buffer E 100 mM NaH2PO4 10 mM Tris 2 mM GSH reduced glutathione 0.2 mM GSSG oxidised glutathione pH 8.0 Buffer F 100 mM NaH2PO4 10 mM Tris pH 8.0 Buffer G 100 mM NaH2PO4 10 mM Tris 300mM Imidazole pH 8.0 Dialysis Buffer (Tris buffered saline pH7.4) 150 mM NaCl 10 mM Tris pH 8.0 |
| Refolding Assay | Crystallography |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | ~20mg/litre culture |
| Purity | ~90-95% |
| Notes | (pQE9 vector derived seqeunce) and mouse MOG extracellualr domain which contains a single Ig-like V type domain (MRGSHHHHHHGS)-GQFRVIGPGYPIRALVGDEAELPCRISPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPEYRGRTE LLKETISEGKVTLRIQNVRFSDEGGYTCFFRDHSYQEEAAMELKVED |