Refolding Record:
| Protein | |
|---|---|
| Protein Name | ADAMTS13 |
| Abbreviated Name | ADAMTS13 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q76LX8 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 1373 |
| Molecular Weight | 148313.0 |
| Pi | 6.72939 |
| Molecular Weight | 148313.0 |
| Disulphides | 3 |
| Full Sequence |
MHHHHHHGKPIPNPLLGLDSTENLTFQIDPFTMMHQRHPRARC PPLCVAGILA CGFLLGCWGP SHFQQSCLQA LEPQAVSSYL
SPGAPLKGRP PSPGFQRQRQ RQRRAAGGIL HLELLVAVGP DVFQAHQEDT ERYVLTNLNI GAELLRDPSL GAQFRVHLVK MVILTEPEGA PNITANLTSS LLSVCGWSQT INPEDDTDPG HADLVLYITR FDLELPDGNR QVRGVTQLGG ACSPTWSCLI TEDTGFDLGV TIAHEIGHSF GLEHDGAPGS GCGPSGHVMA
SDGAAPRAGL AWSPCSRRQL LSLLSANEQC RVAFGPKAVA CTFAREHLDM CQALSCHTDP LDQSSCSRLL VPLLDGTECG VEKWCSKGRC RSLVELTPIA AVHGRWSSWG PRSPCSRSCG GGVVTRRRQC NNPRPAFGGR ACVGADLQAE MCNTQACEKT QLEFMSQQCA RTDGQPLRSS PGGASFYHWG AAVPHSQGDA LCRHMCRAIG ESFIMKRGDS FLDGTRCMPS GPREDGTLSL CVSGSCRTFG CDGRMDSQQV WDRCQVCGGD NSTCSPRKGS FTAGRAREYV TFLTVTPNLT SVYIANHRPL FTHLAVRIGG RYVVAGKMSI SPNTTYPSLL EDGRVEYRVA LTEDRLPRLE EIRIWGPLQE DADIQVYRRY GEEYGNLTRP DITFTYFQPK
PRQAWVWAAV RGPCSVSCGA GLRWVNYSCL DQARKELVET VQCQGSQQPP AWPEACVLEP CPPYWAVGDF GPCSASCGGG LRERPVRCVE AQGSLLKTLP PARCRAGAQQ PAVALETCNP QPCPARWEVS EPSSCTSAGG AGLALENETC VPGADGLEAP VTEGPGSVDE KLPAPEPCVG MSCPPGWGHL DATSAGEKAP
SPWGSIRTGA QAAHVWTPAA GSCSVSCGRG LMELRFLCMD SALRVPVQEE LCGLASKPGS RREVCQAVPC PARWQYKLAA CSVSCGRGVV RRILYCARAH GEDDGEEILL DTQCQGLPRP EPQEACSLEP CPPRWKVMSL GPCSASCGLG TARRSVACVQ LDQGQDVEVD EAACAALVRP EASVPCLIAD CTYRWHVGTW
MECSVSCGDG IQRRRDTCLG PQAQAPVPAD FCQHLPKPVT VRGCWAGPCV GQGACGRQHL EPTGTIDMRG PGQADCAVAI GRPLGEVVTL RVLESSLNCS AGDMLLLWGR LTWRKMCRKL LDMTFSSKTN TLVVRQRCGR PGGGVLLRYG SQLAPETFYR ECDMQLFGPW GEIVSPSLSP ATSNAGGCRL FINVAPHARI
AIHALATNMG AGTEGANASY ILIRDTHSLR TTAFHGQQVL YWESESSQAE MEFSEGFLKA QASLRGQYWT LQSWVPEMQD PQSWKGKEGT
|
| Notes | Carboxyl-terminal truncated domains of ADAMTS13 from E.coli also expressed and refolded: His-V5-Dis, encodes disintegrin domain His-V5-TSP1, Encodes up to first tsp1 (approximately 357-407) His-V5-CysR, encodes up to spacer His-V5-Spa, encodes spacer various c-terminal truncated domains of ADAMTS13 |
| Expression | |
|---|---|
| Report | Ai, J., Smith, P., Wang, S., Zhang, P., and Zheng, X.L. (2005) J Biol Chem, 280, 29428-29434 |
| Project Aim | Structure-Function |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21Ai |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pcDNA3.1 V5-His TOPO |
| Expression Protocol | 1 L of culture medium was inoculated with a single colony. 0.05 mM ITPG was used to induce cells ( 4h at 37degC) before harvesting by centrifugation ( 3,500 rpm for 15 min). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Chemical |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 mM Urea in 100 mM NaH2PO4, 10 mM TrisHCl, pH 8 |
| Refolding Buffer | 50 mM MES, pH 6.0, 240 mM NaCL, 10 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.5 M arginine, 0.4 M sucrose, 0.5% Triton X-100, 0.05% polyethylene glycol 16 000 in 1 mM fresh GSH and 0.1 mM fresh GSSH |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 22.0 |
| Protein Concentration | |
| Refolding Time | 72h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | n/a,n/a,n/a,1mM/0.1mM,n/a |
| Refolding Protocol | The inclusion bodies were isolated using B-Per reagents before re-suspension. The protein was then added in a drop wise manner to the refolding buffer before being dialyzed against 20 mM TrisHCl, 150 mM NaCl, pH 8. The recombinant proteins were then purified via affinity chromatography with nickel-nitrilotriacetic acid-agarose. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine,Polyethylene glycol (PEG) |
| Additives Concentration | 0.5M/0.05% |
| Refolding Yield | |
| Purity | |
| Notes | |