Refolding Record:
| Protein | |
|---|---|
| Protein Name | Coagulation factor VIII |
| Abbreviated Name | FVIII |
| SCOP Family | Coagulation factor VIII, membrane-binding peptide |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P00451 |
| 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 | 2334 |
| Molecular Weight | 264726.0 |
| Pi | 6.96 |
| Molecular Weight | 264726.0 |
| Disulphides | 8 |
| Full Sequence |
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAG
VQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Huang CC, Li LT, Shen MC, Chen JY, Lin SW. (2001) Thrombosis Research, 101, 405-415 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | A total of eight plasmids were constructed to direct expression of FVIII. Details of the construction of these expression plasmids are described elsewhere [24]. FVIII expression plasmids were subcloned into E. coli BL21(DE3)pLysS for the synthesis of FVIII fusion peptides by induction of the bacterial cultures with IPTG, as previously described [24]. The bacteria were harvested and stored at −20°C until use. The soluble fusion peptide was purified by one step metal-ion chelating chromatography with a Ni-NTA column, following the manufacturer\'s instructions (Qiagen, Chatsworth, CA, USA). Insoluble fusion peptides were collected as inclusion bodies in bacterial lysates and were partially purified as previously described [25], with minor modifications. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.5% Triton X-100 and 1 mM EDTA |
| Solubilization Buffer | 3 M Guanidine–HCl or 6M urea |
| Refolding Buffer | appropriate buffers |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 0.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | 3 day |
| Redox Agent | Not specified |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Insoluble fusion peptides were collected as inclusion bodies in bacterial lysates and were partially purified as previously described [25], with minor modifications. Briefly, harvested bacterial pellets were lysed by sonication, washed twice with 0.5% Triton X-100 and 1 mM EDTA, and denatured in 3 M Guanidine–HCl or 6M urea for periods ranging from several hours to overnight. The mixture was dialyzed overnight against appropriate buffers and collected for centrifugation for 30 min at 12,000×g. The supernatant was analyzed by SDS/PAGE (see below) for the presence of soluble FVIII fusion peptides. In the cases of 8A2 and 8A3 fusion peptides, solubilization was performed with the protein bound to a solid support. After denaturation in 8 M urea, 10 mM 2-mercaptoethanol, 0.1 M NaH2PO4 and 10 mM Tris–HCl, pH 8, the mixture was allowed to stand at room temperature overnight. After centrifugation at 12,000×g for 30 min, the supernatant was applied onto the Ni-NTA column to absorb the fusion peptides. The resin with fusion proteins was dialyzed overnight against 1 M urea at different pH levels and allowed to incubate further in the same solution at 10°C for 3 days. The fusion peptides were then eluted from the resin with 250 mM imidazole, 0.1 M NaH2PO4 and 10 mM Tris–HCl, pH 8 and subsequently dialyzed extensively against appropriate buffers. The obtained fusion proteins were used as antigens for the immunization of mice. |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |