Refolding Record:
| Protein | |
|---|---|
| Protein Name | Levansucrase |
| Abbreviated Name | Levansucrase |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Zymomonas mobilis |
| UniProt Accession | Q60114 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 430 |
| Molecular Weight | 46762.0 |
| Pi | 4.64999 |
| Molecular Weight | 46762.0 |
| Disulphides | 1 |
| Full Sequence |
MLNKAGIAEPSLWTRADAMKVHTDDPTATMPTIDYDFPVMTDKYWVWDTWPLRDINGQVV
SFQGWSVIFALVADRTKYGWHNRNDGARIGYFYSRGGSNWIFGGHLLKDGANPRSWEWSG
CTIMAPGTANSVEVFFTSVNDTPSESVPAQCKGYIYADDKSVWFDGFDKVTDLFQADGLY
YADYAENNFWDFRDPHVFINPEDGKTYALFEGNVAMERGTVAVGEEEIGPVPPKTETPDG
ARYCAAAIGIAQALNEARTEWKLLPPLVTAFGVNDQTERPHVVFQNGLTYLFTISHHSTY
ADGLSGPDGVYGFVSENGIFGPYEPLNGSGLVLGNPSSQPYQAYSHYVMTNGLVTSFIDT
IPSSDPNVYRYGGTLAPTIKLELVGHRSFVTEVKGYGYIPPQIEWLAEDESSNSAAALSL
LNK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sunitha K, Chung BH, Jang KH, Song KB, Kim CH, Rhee SK (2000) Protein Expression and Purification, 18, 388-393 |
| 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 | 30.0 |
| Expression Time | 15h |
| Expression Vector | pRZ4 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Other |
| Wash Buffer | 20mM potassium phosphate, 3M urea, pH 6.4 |
| Solubilization Buffer | 20 mM potassium phosphate, 4% Triton X-100, pH 6.4 |
| Refolding Buffer | 20 mM potassium phosphate, 4% Triton X-100, 0.5M L-glycine |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.2mg/ml |
| Refolding Time | 16 |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Levansucrase was expressed as a soluble active form when produced in a shake flask culture of E. coli/pRZ4 (data not shown). This had also been observed in our previous reports (5,16) on the expression of levansucrase in E. coli carrying an inducible expression plasmid. However, when the fed-batch culture was carried out in a bench-scale fermentor to increase the production level, most of the levansucrase was expressed as cytoplasmic inclusion bodies. The inclusion bodies were solubilized at a protein concentration of 200 mg/mL in 20 mM KPi buffer containing 4% Triton X-100 (Sigma, St. Louis, MO). Solubilization was carried out for the first 10 min with vigorous shaking, and the refolding was carried out under the slow air oxidation condition for 16 h at 4°C with gentle shaking. The inclusion body levansucrase was solubilized and renatured in 4% (v/v) Triton X-100, 0.5M L-glycine at pH 6.4. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 85% |
| Purity | many visible bands on SDS |
| Notes | |