Refolding Record:
| Protein | |
|---|---|
| Protein Name | Matriptase also known as Suppressor of tumorigenicity protein 14 |
| Abbreviated Name | n/a |
| SCOP Family | Eukaryotic Proteases |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q9Y5Y6 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 856 |
| Molecular Weight | 94769.7 |
| Pi | 6.11 |
| Molecular Weight | 94769.7 |
| Disulphides | >10 |
| Full Sequence |
MGSDRARKGGGGPKDFGAGLKYNSRHEKVNGLEEGVEFLPVNNVKKVEKHGPGRWVVLAAVLIGLLLVLLGIGFLVWHLQYRDVRVQKVFNGYMRITNENFVDAYENSNSTEFVSLASKVKDALKLLYSGVPFLGPYHKESAVTAFSEGSVIAYYWSEFSIPQHLVEEAERVMAEERVVMLPPRARSLKSFVVTSVVAFPTDSKTVQRTQDNSCSFGLHARGVELMRFTTPGFPDSPYPAHARCQWALRGDADSVLSLTFRSFDLASCDERGSDLVTVYNTLSPMEPHALVQLCGTYPPSYNLTFHSSQNVLLITLITNTERRHPGFEATFFQLPRMSSCGGRLRKAQGTFNSPYYPGHYPPNIDCTWNIEVPNNQHVKVRFKFFYLLEPGVPAGTCPKDYVEINGEKYCGERSQFVVTSNSNKITVRFHSDQSYTDTGFLAEYLSYDSSDPCPGQFTCRTGRCIRKELRCDGWADCTDHSDELNCSCDAGHQFTCKNKFCKPLFWVCDSVNDCGDNSDEQGCSCPAQTFRCSNGKCLSKSQQCNGKDDCGDGSDEASCPKVNVVTCTKHTYRCLNGLCLSKGNPECDGKEDCSDGSDEKDCDCGLRSFTRQARVVGGTDADEGEWPWQVSLHALGQGHICGASLISPNWLVSAAHCYIDDRGFRYSDPTQWTAFLGLHDQSQRSAPGVQERRLKRIISHPFFNDFTFDYDIALLELEKPAEYSSMVRPICLPDASHVFPAGKAIWVTGWGHTQYGGTGALILQKGEIRVINQTTCENLLPQQITPRMMCVGFLSGGVDSCQGDSGGPLSSVEADGRIFQAGVVSWGDGCAQRNKPGVYTRLPLFRDWIKENTGV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kirchhofer D, Peek M, Li W, Stamos J, Eigenbrot C, Kadkhodayan S, Elliott JM, Corpuz RT, Lazarus RA, Moran P. (2003) Biologycal Chemistry, 19, 36341-36349 |
| Project Aim | Undefined |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | 33D3 |
| Expression Temp | 30.0 |
| Expression Time | 24 h |
| Expression Vector | pSTII.MTSP.PD.H8 |
| Expression Protocol | E. coli strain 33D3 (W3110 fhuA (tonA) ptr3 lac Iq lacL8 ompT (nmpc-fepE) degP41 kanR) was transformed with pSTII.MTSP.PD.H8. Single colonies from a LB carbenicillin plate were inoculated into 5 ml of LB medium supplemented with carbenicillin (50 µg/ml) and grown at 30 °C on a culture wheel overnight. The 5-ml culture was diluted into 500 ml of phosphate-limiting medium (46). Carbenicillin was then added to the induction culture to give a concentration of 50 µg/ml, and the culture was grown for 24 h at 30 °C. |
| Method of Induction | Carbenicillin |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 20 mM Tris-HCl, pH 8.6, containing 6 M guanidine HCl |
| Refolding Buffer | 20 mM Tris-Cl, pH 8.6, 0.8 M arginine, 0.3 M NaCl, 20 mM glycine, 1 mM EDTA, and 1 mM cysteine |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no |
| Refolding pH | 8.6 |
| Refolding Temperature | 8.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | Cysteine |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | E. coli pastes from 500-ml shake flask cultures (6–10-g pellets) were resuspended in 10 volumes (w/v) of 20 mM Tris-HCl, pH 8.0, containing 7 M guanidine HCl. Solid sodium sulfite and sodium tetrathionate were added to make final concentrations of 0.1 and 0.02 M, respectively, and the solution was stirred overnight at 4 °C. The solution was clarified by centrifugation and loaded onto a 20-ml Qiagen Ni-NTA metal chelate column equilibrated in 20 mM Tris-HCl, pH 8.6, containing 6 M guanidine HCl. The column was washed with additional buffer containing 50 mM imidazole (Ultrol grade; Calbiochem). The protein was eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein based on SDS-PAGE were pooled and diluted to 50 µg/ml with buffer containing 20 mM Tris-Cl, pH 8.6, 0.8 M arginine, 0.3 M NaCl, 20 mM glycine, 1 mM EDTA, and 1 mM cysteine. The refolding mixture was incubated overnight at 2–8 °C. The protein was subsequently concentrated 20-fold using Vivascience (Edgewood, NY) concentrator (molecular weight cut-off, 10,000) and dialyzed against 50 mM Tris-HCl, pH 8.0, and 0.15 M NaCl. The refolded protein was loaded on a Superdex 75 (Amersham Biosciences) equilibrated with the same buffer. The fractions were analyzed by SDS-PAGE (>95% purity) and enzymatic activity using a chromogenic substrate (see below) and pooled. The matriptase protease domain was also analyzed by N-terminal amino acid sequencing and electrospray mass spectrometry. Protein concentration was determined by quantitative amino acid analysis. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine,Glycine |
| Additives Concentration | 20 mM/0.8 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |