Refolding Record:
| Protein | |
|---|---|
| Protein Name | Lysyl oxidase |
| Abbreviated Name | LOX |
| SCOP Family | Amine oxidase N-terminal region |
| Structure Notes | |
| Organism | yeast (Pichia pastoris) |
| UniProt Accession | Lysyl oxidase |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 789 |
| Molecular Weight | 89691.1 |
| Pi | 4.52 |
| Molecular Weight | 89691.1 |
| Disulphides | Unknown |
| Full Sequence |
MRLTNLLSLTTLVALAVAVPDFYQKREAVSSKEAALLRRDASAECVSNENVEIEAPKTNIWTSLAKEEVQEVLDLLHSTYNITEVTKADFFSNYVLWIETLKPNKTEALTYLDEDGDLPPRNARTVVYFGEGEEGYFEELKVGPLPVSDETTIEPLSFYNTNGKSKLPFEVGHLDRIKSAAKSSFLNKNLNTTIMRDVLEGLIGVPYEDMGCHSAAPQLHDPATGATVDYGTCNINTENDAENLVPTGFFFKFDMTGRDVSQWKMLEYIYNNKVYTSAEELYEAMQKDDFVTLPKIDVDN
LDWTVIQRNDSAPIRHLDDRKSPRLVEPEGRRWAYDGEEEYFSWMDWGFYTSWSRDTGISFYDITFKGERIVYELSLQELIAEYGSDDPFNQHTFYSDISYGVGNRFSLVPGYDCPATAGYFTTDTFEYDEFYNRTLSYCVFENQEDYSLLRHTGASYSAITQNPTLNVRFISTIGNYDYNFLYKFFLDGTLEVSVRAAGYIQAGYWNPETSAPYGLKIHDVLSGSFHDHVLNYKVDLDVGGTKNRASKYVMKDVDVEYPWAPGTVYNTKQIAREVLEKEDFNGINWPENGQGILLIESA
EETNSFGNPRAYNIMPGGGGVHRIVKNSRSGPETQNWARSNLFLTKHKDEELRSSTALNTNALYDPPVNFNAFLDDESLDGEDIVAWVNLGLHHLPNSNDLPNTIFSTAHASFMLTPFNYFDSENSRDTTQQVFYTYDDETEESNWEFYGNDWSSCGLEVPEPNFEDYTYGRGTRINKKMTNSDEVY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fogelgren B, Polgár N, Szauter KM, Ujfaludi Z, Laczkó R, Fong KS, Csiszar K. (2005) Biologycal Chemistry, 280, 24690-24697 |
| Project Aim | Undefined |
| Fusion | N-terminal GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 2 h |
| Expression Vector | pGBKT7-proLOX |
| Expression Protocol | GST Protein Expression and Pull-downs—The plasmid pGEX-4T-1 (Amersham Biosciences) was used to generate expression constructs for various LOX fragments containing an N-terminal GST tag. The pGBKT7-proLOX plasmid was used as a cDNA template for cloning the LOX fragments. Recombinant GST·LOX fusion proteins, corresponding to amino acids 1–417 (proLOX), 169–417 (mature LOX), 1–168 (propeptide), 169–348 (mature LOX minus the CRL domain), and 349–417 (CRL domain), were cloned and transformed into the BL21 strain of Escherichia coli (Stratagene). GST fusion protein expression was induced by adding 0.1 mM isopropyl-1-thio--D-galactopyranoside to growing cultures and shaking for an additional 2 h at 37 °C. |
| 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 | n/a |
| Solubilization Buffer | 8 M urea, 10 mM K2HPO4, 5 mM dithiothreitol, pH 8.2 |
| Refolding Buffer | 10 mM K2HPO4 buffer (pH 8.2) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.2 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Recombinant proteins were extracted from inclusion bodies using a solubilization buffer (8 M urea, 10 mM K2HPO4, 5 mM dithiothreitol, pH 8.2), filtered through a 0.45-µm syringe filter, and refolded with a rapid dilution into 10 mM K2HPO4 buffer (pH 8.2). GST·LOX fusion proteins were captured and purified using glutathione-Sepharose 4B (Amersham Biosciences) and then eluted using the inclusion body solubilization buffer and refolded. Protein concentrations were measured using Bradford reagent (Bio-Rad). |
| Refolding Assay | Actin Binding Activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |