Anaerob Protein oprensning og kinetisk analyse via ilt elektrode til at studere DesB Dioxygenase aktivitet og hæmning
Summary
Her præsenterer vi en protokol for anaerob protein oprensning, anaerob proteinkoncentration og efterfølgende kinetic karakterisering ved hjælp af en ilt elektrode system. Metoden er illustreret ved hjælp af enzymet DesB, en dioxygenase enzym, som er mere stabilt og aktiv, når renset og gemt i en anaerobt miljø.
Abstract
Ilt-følsomme proteiner, herunder de enzymer, der anvender ilt som et substrat, kan have reduceret stabilitet, når renset ved hjælp af traditionelle aerobe rensning metoder. Dette manuskript illustrerer de tekniske detaljer i anaerob rensningsproces, herunder udarbejdelse af buffere og reagenser, metoder til kolonne kromatografi i en handskerummet, og afsaltning af protein før kinetik. Også beskrevet er metoder til at forberede og ved hjælp af en ilt elektrode til at udføre kinetic karakterisering af en ilt-udnytte enzym. Disse metoder er illustreret ved hjælp af dioxygenase enzymet DesB, gallate dioxygenase fra bakterien Sphingobium sp. stamme SYK-6.
Introduction
Enzymer, der udnytter jern eller andre metaller til at aktivere ilt er ofte modtagelige for inaktivering under rensningsprocessen på grund af deres fjernelse fra den reducerende miljø af en celle. Derfor, disse proteiner skal bruges som cellelysater, underkastes ekstern reduktionsmiddel, eller renses anaerobt for at sikre, at de har optimale enzymatisk aktivitet1,2,3,4. For de enzymer, der er er ilt-følsomme (specielt jern-holdige enzymer), udføre alle oprensning og karakterisering trin samtidig opretholde anaerobe forhold nødvendige for fuldt ud at beskrive dem. Dette har ført forskerne at udvikle hele laboratoriet set-ups inden for rammerne af anaerobe kamre for undersøgelser spænder fra protein udtryk gennem krystallografi5,6,7,8 .
Heri, rapporterer vi metoder for anaerob rensning og kinetic karakterisering af enzymet DesB ved hjælp af en ilt elektrode system. DesB er en gallate dioxygenase fra bakterien Sphingobium sp. stamme SYK-6, der er relateret til LigAB, en protocatecuate dioxygenase fra den samme organisme. Begge enzymer tilhører den type II protocatechuate dioxygenase (PCAD) superfamilien, som ikke er blevet grundigt undersøgt til dato9, sandsynligvis til dels på grund af enzymer i denne superfamilien bliver modtagelige for inaktivering når renset ved hjælp af standard aerob protein oprensning metoder. Da nogle af enzymerne, der PCAD vise substrat promiskuitet, mens andre er substrat-specifikke2,10, yderligere er karakterisering af denne superfamilien nødvendige for at identificere specificitet determinanter. Som det er blevet bemærket i flere enzym udgøres11,12,13,14,15, kan små molekyler ændre aktivitet via direkte konkurrencedygtige hæmning eller binding af molekyler til at adskille allosteriske lommer, som forårsager en stigning eller et fald i enzymaktivitet16. Mens kinetik alene ikke kan differentiere bindende placeringen af en modulator, bestemme omfanget af en ændring af aktivitet er vigtigt for at forstå effekterne. Som sådan, metoder til kinetic karakterisering af indfødte DesB aktivitet og dens aktivitet i nærværelse af 4-nitrocatechol (4NC), et stof, der er almindeligt anvendt til at karakterisere og hæmme dioxygenase enzymer2,17, 18, er vist.
DesB er i stand til at nedbryde gallate, en lignin-afledte aromatiske sammensatte, via en extradiol dioxygenase (EDO) reaktion i hvilken ring åbning er katalyseret bruger ilt som en af substrater10,19. Denne enzymatiske reaktion opstår i forbindelse med fordelingen af lignin, en aromatisk heteropolymer fundet i cellens væg af planter. Lignin kan være depolymeriseret, giver en bred vifte af aromatiske forbindelser, kan yderligere opdeles i centrale metabolitter3,20,21,22,23,24 ,25,26,27,28,29,30,31,32,33 . Extradiol dioxygenases (EDO) katalyserer en ring åbne reaktion på dihydroxylated aromatiske forbindelser, hvor kavalergang forekommer grænser op til en metal-koordineret diol; derimod spaltes intradiol dioxygenases analoge aromatiske forbindelser mellem de to hydroxylgrupper (figur 1). Larss, ligesom mange andre metalloenzymer, har en divalent metal center for koordinerende henstå består af en to-histidin, one-carboxylat triad9,34,35. Disse metalloenzymer blive oxideret, gennem enten autoxidation eller mekanisme-baserede inaktivering, der henviser til, at enzymet gengives inaktive2,36,37,38.
I de eksperimentelle procedurer, der beskrives i dette manuskript, udnytter vi DesB, medlem af PCAD superfamilien fra bakterien Sphingobium sp. SYK-6, til at katalysere tilsætning af ilt på tværs af C4-C5-bond af gallate (figur 2A). Regiochemistry af denne spaltning er analog med LigAB, som er en protocatechuate-4,5-dioxygenase (figur 2B). Hidtil, omfatter undersøgelser af denne gallate dioxygenase ingen rapporter om stoffer, der hæmmer DesB10,19,39. Med brug af aerobe rensning metoder udstillet DesB variable aktivitet, mens med anvendelse af anaerob metoder kunne vi konsekvent få protein med reproducerbare aktivitet. De kinetiske undersøgelser beskrevet her viser metoder for anaerob rensning af DesB, kinetic karakterisering af reaktionen fra DesB med gallate, og hæmning af DesB af 4-nitrocatechol (4NC).
Protocol
Representative Results
Discussion
De kritiske trin i at opnå aktiv, renset DesB protein involverer dannelse og opretholde den reducerede henstå aktive site i enzymet. Som sådan, rette ydeevne af induktion, rensning, koncentration og udvanding trin er afgørende for succes at opnå aktive enzym. Inducerende protein udtryk i nærværelse af 1 mM jernholdige ammonium sulfat sikrer at henstå indgår korrekt i det aktive sted på DesB. Denne metode er inspireret af undersøgelser som dem med amidohydrolase metalloenzymer, som ofte kræver tilføjelse af m…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne takke Dr. Camille Keller Wesleyan University for teknisk support. Særlig tak til Professor Lindsay D. Eltis samt Jenna K. Capyk fra University of British Columbia og Christian Whitman fra University of Texas i Austin, for deres rådgivning vedrørende anaerob protein oprensning metoder og brug af en O2 -følsomme elektrode.
Materials
| Isopropyl β-D-1-thiogalactopyranodise | Gold Bio Technologies | I2481C50 | |
| Coomassie Brilliant Blue R-250 | Bio-Rad | 161-0400 | |
| Ammonium persulfate | Bio-Rad | 161-0700 | |
| 30% Acrylamide | Bio-Rad | 161-0158 | |
| N,N'tetramethyl-ethylenediamine | Bio-Rad | 161-0801 | |
| Amylose Resin High Flow | New England Biolabs | E8022S | |
| BL21 (DE3) competent Escherichia coli cells | New England Biolabs | C2527I | |
| L-cysteine | Sigma Aldrich | C7352 | |
| gallic acid | Sigma Aldrich | G7384 | |
| 4-nitrocatechol | Sigma Aldrich | N15553 | |
| Ferrous ammonium sulfate | Mallinckrodt | 5064 | |
| Sodium dithionite | Alfa Aesar | 33381-22 | |
| wheaton serum bottles | Fisher Scientific | 06-406G | |
| 25 mm Acrodisc PF Syringe Filter with Supor Membrane | Pall Corportation | 4187 | |
| 400 mL Amicon Stirred Cell Concentrator | EMD Millipore | UFSC40001 | |
| 76 mm Millipore Ultracel 10 kDa cutoff reconsituted cellulose membrane filter | EMD Millipore | PLGC07610 | |
| DL-dithiothreitol | Gold Bio Technologies | DTT50 | |
| Sephadex G-25 coarse desalting gal column | GE Healthcare | 17-0033-01 | |
| 2 mL Crimp-Top Vials | Fisher Scientific | 03-391-38 | |
| Oxygraph Plus Electrode Control Unit | Hansatech Instruments | OXYG1 Plus | |
| Oxygen Eletrode Chamber | Hansatech Instruments | DW1 | |
| Electrode Disc | Hansatech Instruments | S1 | |
| PTFE (0.0125 mmX25mm) 30m reel | Hansatech Instruments | S4 | |
| Electrode cleaning Kit | Hansatech Instruments | S16 | |
| Spacer paper | Zig Zag | available at any gas station | |
| He-series Dri-Lab glove box | Vacuum/Atmospheres Company | ||
| HE-493 Dri-Train | Vacuum/Atmospheres Company | ||
| Double-Ended Micro-Tapered Stainless Steel Spatula | Fisher Scientific | 21-401-10 | |
| DWK Life Sciences Kimble Kontes Flex Column Economy Column | Fisher Scientific | k420400-1530 | |
| 10 μL, Model 701 N SYR, Cemented NDL 26s ga, 2 in, point stlye 2 syringe | Hamilton | 80300 | |
| DWK Life Sciences Kimble Kontes Flex Column Economy Column | Fisher Scientific | K420401-1505 | |
| Emulsiflex-C5 high-pressure homogenizer | Avestin | ||
| B-PER Complete Bacterial Protein Extraction Reagent | Thermo Fisher Scientific | 89821 | |
| Lysozyme from chicken egg white | Sigma Aldrich | 12650-88-3 | |
| Sodium dodecyl sulfate | Thermo Fisher Scientific | 151-21-3 | |
| ampicillin | Sigma Aldrich | 7177-48-2 | |
| Tryptone | Fisher Scientific | BP-1421-500 | |
| Yeast extract | Fisher Scientific | BP1422-2 | |
| Sodium Chloride | Fisher Scientific | S271-10 | |
| Potassium Chloride | Fisher Scientific | P217-3 | |
| Magnesium Chloride | Fisher Scientific | M33-500 | |
| Dextrose | Fisher Scientific | D16-3 | |
| Sodium Hydroxide | Fisher Scientific | S318-1 | |
| Tris hydrochloride | Fisher Scientific | BP153-500 | |
| Maltose | Fisher Scientific | BP684-500 | |
| Glycine | Fisher Scientific | G46-500 |
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