Refolding Record:
| Protein | |
|---|---|
| Protein Name | CD4 |
| Abbreviated Name | CD4 |
| SCOP Family | Small Kunitz-type inhibitors & BPTI-like toxins |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P01730 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa.1-107 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 107 |
| Molecular Weight | 12078.7 |
| Pi | 8.58781 |
| Molecular Weight | 12078.7 |
| Disulphides | 3 |
| Full Sequence |
KKVVLGKKGD TVELTCTASQ KKSIQFHWKN SNQIKILGNQ GSFLTKGPSK LNDRADSRRS LWDQGNFPLI IKNLKIEDSD TYICEVEDQK EEVQLLVFGL TANSDTH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Chao BH, Costopoulos DS, Curiel T, Bertonis JM, Chisholm P, Williams C, Schooley RT, Rosa JJ, Fisher RA, Maraganore JM (1989) J Biol Chem, 264, 5812-5817 |
| 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 | lon htpr mutant |
| Expression Temp | 37.0 |
| Expression Time | 4.5h |
| Expression Vector | pBG211.11 |
| Expression Protocol | Cells were diluted into 40ml minimal medium plus tryptophan at 30degC and shaken for 4.5h. Following this dilution, 1ml aliquots were removed into induction medium at 0,2 and 4 h and after overnight growth. |
| Method of Induction | Autoinduction |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20mM Tris pH 7.5, 7M urea, 10mM 2-mercaptoethanol |
| Refolding Buffer | 1: 3M urea, 20mM Tris pH 7.5, 2: 1M urea, 0.1M ammonium acetate pH 6.8, 3: phosphate-buffered saline |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.5 A280/ml |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Wet cells (14g) from a 4L culture were suspended in 100ml of 20mM Tris pH 7.5 containing 20microg/ml DNase, 20microg/ml RNase and 1mM PMSF. The suspension was twice passed through a French press at 1000psi and then centrifuged (15min, 4degC, 18000g). The pellet was then dissolved in 20ml solubilization buffer, then centrifuged (90min, 4degC, 85000g). The supernatant was then diluted with 80ml of solubilization buffer and then applied to a Q-sepharose fast-flow column equilibrated in the same buffer. The protein was eluted from the column with a gradient of 0-0.3M NaCl in solubilization buffer. Pooled fractions containing 20mg protein in 50ml were concentrated to 10ml, then 5ml was applied to a S-300 column equilibrated in solubilization buffer. The protein was refolded by step-wise dialysis against 500 volumes of Refolding buffer 1, followed by 500 volumes of refolding buffer 2, followed by 500 volumes of refolding buffer 3. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |