Refolding Record:
| Protein | |
|---|---|
| Protein Name | Glucoamylase |
| Abbreviated Name | Glucoamylase |
| SCOP Family | Glucoamylase |
| Structure Notes | |
| Organism | Saccharomycopsis fibuligera |
| UniProt Accession | Q8TFE5 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 523 |
| Molecular Weight | 57422.9 |
| Pi | 5.78849 |
| Molecular Weight | 57422.9 |
| Disulphides | 0 |
| Full Sequence |
MIRLTVFLTAVFAAVASCVPVELDKRNTGHFQAYSGYTVARSNFTQWIHEQPAVSWYYLL
QNIDYPEGQFKSAKPGVVVASPSTSEPDYFYQWTRDTAITFLSLIAEVEDHSFSNTTLAK
VVEYYISNTYTLQRVSNPSGNFDSPNHDGLGEPKFNVDDTAYTASWGRPQNDGPALRAYA
ISRYLNAVAKHNNGKLLLAGQNGIPYSSASDIYWKIIKPDLQHVSTHWSTSGFDLWEENQ
GTHFFTALVQLKALSYGIPLSKTYNDPGFTSWLEKQKDALNSYINSSGFVNSGKKHIVES
PQLSSRGGLDSATYIAALITHDIGDDDTYTPFNVDNSYVLNSLYYLLVDNKNRYKINGNY
KAGAAVGRYPEDVYNGVGTSEGNPWQLATAYAGQTFYTLAYNSLKNKKNLVIEKLNYDLY
NSFIADLSKIDSSYASKDSLTLTYGSDNYKNVIKSLLQFGDSFLKVLLDHIDDNGQLTEE
INRYTGFQAGAVSLTWSSGSLLSANRARNKLIELL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Solovicova A, Gasperik J, Hostinova E. (1996) Biochemical and Biophysical Research Com, 224, 790-795 |
| 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 | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 8h |
| Expression Vector | pET3d |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 20mM NaCl, 1mM DTE, 1mM EDTA, 0.5mM PMSF, pH 7.5 |
| Solubilization Buffer | 8M urea, pH 7.0 |
| Refolding Buffer | 50mM Tris-HCl, 20% glycerol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 7.2 |
| Refolding Temperature | 254.0 |
| Protein Concentration | |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The solubilised protein was diluted 100-fold in 50mM Tris-HCl, 20% glycerol pH 7.2. The solution was then dialysed continually in an artificial kidney in a cold room against 6L of the same buffer. The protein solution was stored at 4 degrees C for 24h prior to purification with anion exchange chromatography. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 30mg/L culture |
| Purity | |
| Notes | |