Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alternansucrase |
| Abbreviated Name | ASR |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Leuconostoc mesenteroides |
| UniProt Accession | Q9RE05 |
| 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 | 2058 |
| Molecular Weight | 228850.0 |
| Pi | 5.31 |
| Molecular Weight | 228850.0 |
| Disulphides | Unknown |
| Full Sequence |
MKQQETVTRKKLYKSGKVWVAAATAFAVLGVSTVTTVHADTNSNVAVKQINNTGTNDSGEKKVPVPSTNNDSLKQGTDGFWYDSDGNRVDQKTNQILLTAEQLKKNNEKNLSVISDDTSKKDDENISKQTKIANQQTVDTAKGLTTSNLSDPITGGHYENHNGYFVYIDASGKQVTGLQNIDGNLQYFDDNGYQVKGSFRDVNGKHIYFDSVTGKASSNVDIVNGKAQGYDAQGNQLKKS
YVADSSGQTYYFDGNGQPLIGLQTIDGNLQYFNQQGVQIKGGFQDVNNKRIYFAPNTGNAVANTEIINGKLQGRDANGNQVKNAFSKDVAGNTFYFDANGVMLTGLQTISGKTYYLDEQGLRKNYAGTFNNQFMYFDADTGAGKTAIEYQFDQGLVSQSNENTPHNAAKSYDKSSFENVDGYLTADTWYRPTDILKNGDTWTASTETDMRPLLMTWWPDKQTQANYLNFMSSKGLGITTTYTAATSQKTLNDAAFVIQTAIEQQISLKKSTEWLRDAIDSFVKTQANWNKQTEDEAFDGLQWLQGGFLAYQDDSHRTPNTDSGNNRKLGRQPINIDGSKDTTDGKGSEFLLANDIDNSNPIVQAEQLNWLHYLMNFGSITGNNDNANFDGIRVDAVDNVDADLLKIAGDYFKALYGTDKSDANANKHLSILEDWNGKDPQYVNQQGNAQLTMDYTVTSQFGNSLTHGANNRSNMWYFLD
TGYYLNGDLNKKIVDKNRPNSGTLVNRIANSGDTKVIPNYSFVRAHDYDAQDPIRKAMIDHGIIKNMQDTFTFDQLAQGMEFYYKDQENPSGFKKYNDYNLPSAYAMLLTNKDTVPRVYYGDMYLEGGQYMEKGTIYNPVISALLKARIKYVSGGQTMATDSSGKDLKDGETDLLTSVRFGKGIMTSDQTTTQDNSQDYKNQGIGVIVGNNPDLKLNNDKTITLHMGKAHKNQLYRALVLSNDSGIDVYDSDDKAPTLRTNDNGDLIFHKTNTFVKQDGTIINYEMKGSLNALISGYLGVWVPVGASDSQDARTVATESSSSNDGSVFHSNAALDSNVIYEGFSNFQAMPTSPEQSTNVVIATKANLFKELGITSFELAPQYRSSGDTNYGGMSFLDSFLNNGYAFTDRYDLGFNKADGNPNPTKYGTDQDLRNAIEALHKNGMQAIADWVPDQIYALPGKEVVTATRVDERGNQLKDTDFVNLLYVANTKSSGVDYQAKYGGEFLDKLREEYPSLFKQNQVSTGQPIDASTKIKQWSAKYMNGTNILHRGAYYVLKDWATNQYFNIAKTNEVFLPLQLQNKDAQTGFISDASGVKYYSISGYQAKDTFIEDGNGNWYYFDKDGYMVRSQQGENPIRTVETSVNTRNGNYYFMPNGVELRKGFGTDNSGNVYYFDDQGKMVRDKYINDDANNFYHLNVDGTMSRGLFKFDSDTLQYFASNGVQIKDSYAKDSKGNKYYFDSATGNNDTGKAQTWDGNGYYITIDSDANNTIGVNTDYTAYITSSLREDGLFANAPYGVVTKDQNGNDLKWQYINHTKQYEGQQVQVTRQYTDSKGVSWNLITFAGGDLQGQRLWVDSRALTMTPFKTMNQISFISYANRNDGLFLNAPYQVKGYQLAGMSNQYKGQQVTIAGVANVSGKDWSLISFNGTQYWIDSQALNTNFTHDMNQKVFVNTTSNLDGLFLNAPYRQPGYKLAGLAKNYNNQTVTVSQQYFDDQGTVWSQVVLGGQTVWVDNHALAQMQVSDTDQQLYVNSNGRNDGLFLNAPYRGQGSQLIGMTADYNGQHVQVTKQGQDAYGAQWRLITLNNQQVWVDSRALSTTIMQAMNDNMYVNSSQRTDGLWLNAPYTMSGAKWAGDTRSANGRYVHISKAYSNEVGNTYYLTNLNGQSTWIDKRAFTVTFDQVVALNATIVARQRPDGMFKTAPYGEAGAQFVDYVTNYNQQTVPVTKQHSDAQGNQWYLATVNGTQYWIDQRSFSPVVTKVVDYQAKIVPRTTRDGVFSGAPYGEVNAKLVNMATAYQNQVVHATGEYTNASGITWSQFALSGQEDKLWIDKRALQA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Joucla G, Pizzut S, Monsan P, Remaud-Simeon M. (2006) FEBS Letters, 580, 763-768 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 15 h |
| Expression Vector | n/a |
| Expression Protocol | E. coli One Shot TOP10 (Invitrogen) was used to transform the constructed plasmids and to express the truncated asr genes. Bacterial cells were grown on LB medium with 100 μg ml−1 of ampicillin. The induction was performed using 0.02% arabinose (w/v) at OD600 nm of 0.6. After 15 h following the induction (OD600 nm about 2.5 or 3.9 g l−1 of biomass) cells were harvested by centrifugation (4500 × g, 10 min, 4 °C) and resuspended in lysis buffer (20 mM sodium acetate buffer, pH 5.4; Triton X-100, 1%; lysozyme, 1 mg ml−1; DNaseI, 5 mg ml−1) to an optical density OD600 nm of 80. |
| Method of Induction | Arabinose |
| Cell Density at Induction | OD 0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | urea 8 M, pH 8.0 |
| Refolding Buffer | sodium acetate buffer 20 mM, pH 5.4 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 5.4 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were disrupted by sonication. The protein extracts obtained were centrifuged (27 000 × g, 30 min, 4 °C). The supernatants were tested for protein content resulting in concentration of 12 ± 1 g l−1 and were tested for activity. The amount of recovered activity was named soluble activity and expressed in U l−1 of culture. The pellet of sonication was resuspended and tested for activity. It was named insoluble activity and was also expressed in U l−1 of culture. Renaturation of the pellet was also tested by 1 h incubation in urea 8 M, pH 8.0, followed by overnight dialysis against sodium acetate buffer 20 mM, pH 5.4. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | The temperature is not stated |