Refolding Record:
| Protein | |
|---|---|
| Protein Name | Corrinoid/iron-sulfur protein |
| Abbreviated Name | C/Fe-SP |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Moorella thermoacetica (Clostridium thermoaceticum |
| UniProt Accession | Q07340 |
| 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 | 768 |
| Molecular Weight | 82943.0 |
| Pi | 5.16 |
| Molecular Weight | 82943.0 |
| Disulphides | Unknown |
| Full Sequence |
PLTGLEIYKQLPKKNCGECGTPTCLAFAMNLASGKASLDSCPYVSDAAREALDAAAAPPIAKVVLGAGPTAVEMGDETELFRHDKRFYHETAIAIQVSDNLSSEELKAKVEAINGLNFDRVGQHYTIQAIAIRHDADDPAAFKAAVASVAAATQLNLVLMADDPDVLKEALAGVADRKPLLYAATGANYEAMTALAKENNCPLAVYGNGLEELAELVDKIVALGHKQLVLDPGARETSRAIADFTQIRRLAIKKRFRSFGYPIIALTTAANPLDEVLQAVNYVTKYASLVVLRTDAKEH
LLPLLSWRQNLYTDPQVPIRVEEKLNEIGAVNENSPVYVTTNFSLTYYSVEGEIESTKIPSYLLSVDTDGLSVLTAYADGKFEAEKIAAVMKKVDLDNKVKRHRIIIPGAVAVLKGKLEDLTGWEVIVGPREASGIVAFARANLASAVQILRDRSRAAVQKVVLGATKDQGGTRSHTIVVGGDAALPFHHFEGEIVNRPVIGMEVQDIVPDWPDVLKDPFTDVINEPGRWAQKCVAEYGADLIYLKLDGADPEGANHSVDQCVATVKEVLQAVGVPLVVVGCGDVEKDHEVLEAVAEAAAGENLLLGNAEQENYKSLTAACMVHKHNIIARSPLDINICKQLNILINEMNLPLDHIVIDPSIGGLGYGIEYSFSIMERIRLGALQGDKMLSMPVICTVGYEAWRAKEASAPVSEYPGWGKETERGILWEAVTATALLQAGAHILLMRHPEAVARVKENIDQLMVSNAY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lu WP, Schiau I, Cunningham JR, Ragsdale SW. (1993) Biologycal Chemistry, 15, 5605-5614 |
| Project Aim | Recombinant Protein Expression |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET946B55 |
| Expression Protocol | Overexpression of the C/Fe-SP Genes-Overexpression of acsC was accomplished by insertion of the acsC-D region into a PET plasmid behind a T7 RNA polymerase promoter as depicted in Fig. 1. Plasmid pCt946B (8) (Fig. 2) was digested with BamHI and Hind111 and inserted into the BamHI/HindIII sites of pBluescript KS M13- to yield pBS946B. Using the polymerase chain reaction (PCR) technique to mutate this gene, a 1-kb PCR fragment was generated with pBS946B as the template. The two primers used were: a 21-base 3\' primer complementary to bases 1026-1047 downstream of the initiation codon of the large subunit, and a &-base 5\' mutagenesis primer, TAATCATGAAAGAAAGGAGctcACAcATATGCCTTTGAC. In the latter primer, an NdeI site (underlined) coinciding with the initiation ATG codon for the large subunit, and a Sac1 site (underlined) were included. The mutated bases are shown as lowercase letters. A 175-base pair SacI-Not1 subfragment was inserted into pBS946B to generate the mutant, pBS946B55. The Not1 site is located 160 bases downstream of the initiation codon of the large subunit gene. The sequence of the 175-base PCR-generated insert was verified by DNA sequence analysis. Finally, the entire gene encoding the C/Fe-SP was inserted as an NdeI-Hind111 fragment of pBS946B55 into the NdeIIHindIII sites of PET3a (19) to generate pET946B55. Plasmid pET946B55 was transformed (13) into the expression strain BL21(DE1) from Novagen (19), which was grown aerobically on LB-ampicillin medium at 37 \"C. To induce production of the C/Fe-SP, 0.4 mM IPTG was added during the log phase of coli strain BLZl(DE1) (pET946B55) cells were centrifuged, susgrowth. pended in Buffer A (50 mM Tris-HC1, 5 mM dithiothreitol, pH 7.6), Overexpression of acsD was achieved by subcloning a ClaI-Hind111 and lysed using a Heat Systems XL sonicator inside of a Coy anaerfragment from pCt946B (8) into pBluescript vector KS M13-, giving obic chamber. Centrifugation of the lysate at a setting of 4 (-1500 X pSCP, (Fig. 2) and transformed into E. coli strain HB101. g) for 30 min in an IEC Clinical centrifuge yielded a turbid cell-free Reconstitution and Purification of the Recombinant C/Fe-SP-Four supernatant. This supernatant was then loaded anaerobically into hours after addition of IPTG, when the Awn,,, had reached -2.0, E. capped stainless steel centrifuge tubes and centrifuged in a type 35 rotor in a Beckman L8-55M ultracentrifuge at 30,000 rpm for 1 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris-HC1, 5 mM dithiothreitol, pH 7. |
| Solubilization Buffer | 50 mM Tris-HC1, 5 mM dithiothreitol, pH 7, 6 M urea |
| Refolding Buffer | 50 mM Tris-HC1, 5 mM dithiothreitol, pH 7, hydroxocobalamin 5 umol, 4 M urea |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 13.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | The pellet fraction, which consists mostly of inclusion bodies, was solubilized in Buffer A containing -4 M urea. All steps of the reconstitution were performed at 13 “C in the anaerobic chamber. The inclusion body fraction (-500 mg of protein) was solubilized in 6 M urea in a final volume of -15ml. Then, ydroxocobalamin (5 pmol) was added and, 15 min later, -250 ml of Buffer A was added to dilute the urea concentration to below 0.4 M. After incubation for a few hours, the solution was concentrated in an Amicon ultrafiltration unit with a YM30 membrane to -5 ml and centrifuged to remove precipitated protein. Unbound cobalamin was removed either by repeated buffer exchange with a Minicon B15 concentrator or by centrifugation through a Sephadex G-50 Penefsky spun column (20). |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Please do not submit this it is not finished yet |