Refolding Record:
| Protein | |
|---|---|
| Protein Name | Large structural protein |
| Abbreviated Name | protein L |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Sendai virus (strain Fushimi) |
| UniProt Accession | Q06996 |
| 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 | 2230 |
| Molecular Weight | 252512.0 |
| Pi | 6.36 |
| Molecular Weight | 252512.0 |
| Disulphides | Unknown |
| Full Sequence |
MDGQESSQNPSDILYPECHLNSPIVRGKIAQLHVLLDVNQPYRLKDDSIINITKHKIRNGGLSPRQIKIRSLGKALQRTIKDLDRYTFDPYPTYSQELLRLDIPEICDKIRSVFAVSDRLTRLSSGFQDLWLNIFKQLGNIEGREGYDPLQDISTIPEITDKYSRNRWYRPFLTWFSIKYDRWMQKTRPGGPLDTSNSHNLLECKSYTLVTYGDLVMILNKLTLTGYILTPELVLMYCDVVEGRWNMSAAGHLDKRSIGITSKGEELWELVDSLFSSLGEEIYNVIALLEPLSLALIQLNDPVIPLRGAFMRHVLTELQTVLTSRDVYTDAEADTIVESLLAIFHGTSIDEKAEIFSFFRTFGHPSLEAVTAADKVRAHMYAQKAIKLKTLYECHAVFCTIIINGYRERHGGQWPPCDFPDHVCLELRNAQGSNTAISYECAVDNYTSFIGFKFRKFIEPQLDEDLTIYMKDKALSPRKEAWDSVYPDSNLYYKAPESEETRRLIEVFINDENFNPEEIINYVESGDWLKDEKFNISYSLKEKEIKQEGRLFAKMTYKMRAVQVLAETLLAKGIGELFSENGMVKGEIDLLKRLTTLSVSGVPRTDSVYNNSKSSEKRNEGMKKKNSGGYWDEKKRSRHEFKATDSSTDGYETLSCFLTTDLKKYCLNWRFESTALFGQRCNEIFGFKTFFNWMHPVLERCTIYVGDPYCPVADRMHRQLQDHADSGIFIHNPRGGIEGYCQKLWTLISISAIHLAAVRVGVRVSAMVQGDNQAIAVTSRVPVAQTYKQKKNHVYEETTKYFGALRHVMFDVGHELKLNETIISSKMFVYSKRIYYDGKILPQCLKALTRCVFWSETLVDENRSACSNISTSIAKAIENGYSPILGYCIALYKTCQQVCISLGMTINPTISPTVRDQYFKGKNWLRCAVLIPANVGGFNYMSTSRCFVRNIGDPAVAALADLKRFIRADLLDKQVLYRVMNQEPGDSSFLDWASDPYSCNLPHSQSITTIIKNITARS
VLQESPNPLLSGLFTETSGEEDLNLASFLMDRKVILPRVAHEILGNSLTGVREAIAGMLDTTKSLVRASVRKGGLSYGILRRLVNYDLLQYETLTRTLRKPVKDNIEYEYMCSVELAVGLRQKMWIHLTYGRPIHGLETPDPLELLRGTFIEGSEVCKLCRSEGADPIYTWFYLPDNIDLDTLTNGSPAIRIPYFGSATDERSEAQLGYVRNLSKPAKAAIRIAMVYTWAYGTDEISWMEAALIAQTRANLSLENLKLLTPVSTSTNLSHRLKDTATQMKFSSATLVRASRFITISNDNMALKEAGESKDTNLVYQQIMLTGLSLFEFNMRYKKGSLGKPLILHLHLNNGCCIMESPQEANIPPRSTLDLEITQENNKLIYDPDPLKDVDLELFSKVRDVVHTVDMTYWSDDEVIRATSICTAMTIADTMSQLDRDNLKEMIALVNDDDVNSLITEFMVIDVPLFCSTFGGILVNQFAYSLYGLNIRGREEIWGHVVRILKDTSHAVLKVLSNALSHPKIFKRFWNAGVVEPVYGPNLSNQDKILLALSVCEYSVDLFMHDWQGGVPLEIFICDNDPDVADMRRSSFLARHLAYLCSLAEISRDGPRLESMNSLERLESLKSYLELTFLDDPVLRYSQLTGLVIKVFPSTLTYIRKSSIK
VLRTRGIGVPEVLEDWDPEADNALLDGIAAEIQQNIPLGHQTRAPFWGLRVSKSQVLRLRGYKEITRGEIGRSGVGLTLPFDGRYLSHQLRLFGINSTSCLKALELTYLLSPLVDKDKDRLYLGEGAGAMLSCYDATLGPCINYYNSGVYSCDVNGQRELNIYPAEVALVGKKLNNVTSLGQRVKVLFNGNPGSTWIGNDECEALIWNELQNSSIGLVHCDMEGGDHKDDQVVLHEHYSVIRIAYLVGDRDVVLISKIAPRLGTDWTRQLSLYLRYWDEVNLIVLKTSNPASTEMYLLSR
HPKSDIIEDSKTVLASLLPLSKEDSIKIEKWILIEKAKAHEWVTRELREGSSSSGMLRPYHQALQTFGFEPNLYKLSRDFLSTMNIADTHNCMIAFNRVLQDTIFEWARITESDKRLKLTGKYDLYPVRDSGKLKTISRRLVLSWISLSMSTRLVTGSFPDQKFEARLQLGIVSLSSREIRNLRVITKTLLDRFEDIIHSITYRFLTKEIKILMKILGAVKMFGASQNEYTTVIDDGSLGDIEPYDSS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Einberger H, Mertz R, Hofschneider PH, Neubert WJ. (1990) J Virol, 64, 4274-4280 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein synthesized by chemical means (not recombinant) and refolded. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 100 ml of 0.01 M sodium phosphate (pH 6.8)-3% SDS (BHD Chemicals Ltd., Poole, England)-0.01 M dithio- |
| Solubilization Buffer | 0.01 M sodium phosphate, pH 6.8, containing 0.1% SDS-0.02% NaN3-0.002 M DTT, and the proteins were eluted with a linear gradient of 0.01 to 0.5 M sod |
| Refolding Buffer | 0.1 M Tris hydrochloride [pH 8.5], 0.04 M MgCl2, 0.002 M DTT) and subsequently diluted with one volume of 0.02 M DTT. |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 2 h |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.02 mM |
| Refolding Protocol | Four volumes of cold acetone (-20°C) were added to the SDS-protein solution, and the samples were allowed to precipitate for 30 min in a dry ice-ethanol bath. The tubes then were centrifuged for 10 min at 12,000 x g, the acetone supernatant was poured off, and finally the tubes were inverted to drain. The acetone precipitate was allowed to dry for about 10 min and completely dissolved in 20 RI of dilution buffer (8 M guanadine hydrochloride, 0.1 M Tris hydrochloride [pH 8.5], 0.04 M MgCl2, 0.01 M DTT, 0.04% Nonidet P-40). For renaturation, the samples were dialyzed for 2 h at room temperature and for 12 to 16 h at 4°C against renaturation buffer (0.1 M Tris hydrochloride [pH 8.5], 0.04 M MgCl2, 0.002 M DTT) and subsequently diluted with one volume of 0.02 M DTT. |
| Refolding Assay | Protein kinase assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |