Name: pcr mutagenesis, cloning, expression, fast protein purification protocols and crystallization of the wild type and mutant forms of tryptophan synthase
Uploaded: 2020-09-26T15:00:00
Description: Watch this Scientific Journal Video about PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase at JoVE

Dette arbejde blev støttet af US National Institute of Health (GM097569).

1. Hurtig protokol til rensning af α- og β-underenhed og rekombineret α2β2StTS-kompleks
<ol><li>DNA-subklonering i pETSUMO-udtryksvektor<ol><li>Anskaffelse af det translationelt koblingsgen (trpA og trpB), der kodper α- og β- underenheder af tryptophansyntase fra bakterien Salmonella enterica serovar typhimurium klonet i pEBA-10-udtryksvektoren22. Brug pEBA-10 vektor som DNA-skabelon. BEMÆRK: Alternativt kan de anførte...

<ol>
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Vi har med succes konstrueret mutantform α2β2 βQ114A, α2β2 βK167T og α2β2 βS377A StTS-komplekser til strukturfunktionskorrelationsundersøgelser. I første omgang har vi forsøgt at rense mutanterne ved hjælp af en tidligere rensningsprotokol22, som kræver α2β2St TSkompleks krystallisering med PEG 8000 og spermin under rensning. Selvom krystalliseringshastigheden afhænger af muta...

Tryptofan synthase (TS) bienzym kompleks (α2β2)er et allosterisk enzym, katalysere de sidste to trin i biosyntesen af aminosyren L-Tryptophan i bakterier, planter og svampe1,2,3. Bakterie Salmonella enterica serovar typhimurium (St) forårsager en alvorlig mave-tarminfektion hos mennesker og andre dyr. Da mennesker og højere dyr ikke har TS (EF 4.2.1.20), hæmning af S. typhimurium α2β2 TS-kompleks (α2β2StTS) er blevet undersøgt som et potentielt lægemiddelmål til behandling af cryptosporidiose og tuberkulose4, genitale og okulære infektioner5og til potentiel herbicidudnyttelse i landbruget6. Den α-subunit katalyserer den aldolytiske kavalergang af indol-3-glycerol-fosfat (IGP) til glyceraldehyd-3-fosfat (GAP) og inddol, gennem dannelsen af en indolenine tautomer mellemliggende og efterfølgende kulstof-carbon bond kavalergang til at producere GAP og indole3,6. Det β-katalytiske sted indeholder et pyridoxalt 5′-fosfat (PLP) cofaktormolekyle bundet til β-Lys87 via en Schiff-base, der fungerer som en elektronvask i løbet af reaktionerne på enzymet β-subunit3,7. Den β-site katalyserer udskiftningen af L-Serine sidekæde hydroxyl med inddol for at give L-Tryptophan og et vandmolekyle i en PLP-afhængig reaktion. St. TS fungerer som en mangeårig model for undersøgelse af substratkanalisering og allosterisk kommunikation inden for multienzymkomplekser2,3. Tovejs allosterisk kommunikation mellem TS's α- og β-underenheder er nødvendig for at synkronisere de katalytiske trin og forhindre frigørelse af indoler under L-Tryptophan-syntese3. For at udvide denne indsats har vi forberedt flere mutanter (β-Gln114Ala, β-Lys167Thr og β-Ser377Ala) ved enkeltpunktsmutation, der skal bruges til yderligere udforskning af forholdet mellem enzymstruktur, mekanisme og funktion på det katalytiske sted i StTS β-subunit.
Detaljeret forskning om katalysatormekanismen for α2β2StTS blev indledt af forskningsgruppen Edith W. Miles. Tidlige undersøgelser med indfødte Escherichia coli α2β2 TS kompleks har fokuseret på rensning og karakterisering af den isolerede α-subunit8,9, isolerede β-subunit10,11 og rekonstituering af α2β2 TS kompleks fra de isolerede underenheder12. Rensningen blev udført ved ammoniumsulfatudfældning, prøvedialyse, DEAE-Sephadex-kromatografi, dialyse og en anden kromatografisk runde på en DEAE-Sephadex kolonne12. I en anden protokol blev rensningen af det samme kompleks forbedret ved at indlæse det afklarede celle lysat på en DEAE-Sephadex kolonne efterfulgt af et kromatografisk trin på en Sepharose 4B kolonne, ammonium sulfatudfældning og dialyse13. Begge renselsesprotokoller varer i 4-5 dage. Escherichia coli α2β2 TS kompleks krystalliseret, men krystaller var ikke egnet til X-ray diffraktion på det tidspunkt.
I en ny undersøgelse, rekombinant og vilde type former for S. typhimurium α2β2 TS kompleks blev renset og krystalliseret14,15. Den rekombinant α2β2StTS kompleks var overekspresseret i E. coli stamme CB149 transporterer pEBA-10 udtryk vektor. Indledende krystallisering og X-ray diffraktion dataindsamling og analyse af α2β2StTS kompleks blev rapporteret14. Men, lange og tynde nål som α2β 2StTSkrystallernedsat strukturelle undersøgelser. I et forsøg på at indsamle bedre røntgen diffraktionsdata blev en anden rensningsprotokol beskrevet for at rense den vilde type og mutante former af α2β2St TS-kompleks15. Rensning blev udført med en indledende nedbør ved hjælp af spermin og PEG 8.000 i det afklarede celle lysat, og et stort voluminøst bundfald blev fjernet ved centrifugering. Den supernatantfraktion, der indeholder store mængder α2β2St.TS-kompleks, blev opbevaret i 16-48 timer ved 4 °C, indtil gule krystaller udfældede. Krystaller blev vasket og grundigt dialyzed mod forskellige buffere. Proteinkompleks blev recrystallized i buffer indeholdende ammonium sulfat og dialyzed15. Selvom proteinkrystallisering afhænger af protein- og præcipitantkoncentrationer i opløsning, er det vanskeligt at overvåge, forudsige og reproducere rensning for andre mutante former for α2β2StTS-kompleks i opløsning. Denne protokol har den fordel, at den ikke bruger kromatografiske metoder. Ulemperne er dog den lange rensningstid, der er nødvendig for at krystallisere, dialyse og rekrystallisere, hvilket typisk kræver 5-7 dage. For at opnå krystaller, der er egnede til indsamling af røntgendata, blev mere end 600 krystalliseringsbetingelser evalueret ved hjælp af en kombination og variation af proteinkoncentration, temperatur, præcipitanter (PEG 4.000, 6.000 og 8.000) og tilsætningsstoffer (CaCl2, MnCl2, ZnCl2, kadaver, putrescine, spermine eller spermidin)15. Krystaller havde en bedre krystallinsk form og voksede hurtigere under forhold, der indeholder 12% PEG 8.000 og 2 mM spermin. Krystalliseringen var gunstigere ved 25 °C i stedet for ved 4, 30 eller 42 °C og voksede til maksimale dimensioner inden for 3 dage15. Flere α2β2StTS krystal strukturer blev rapporteret på det tidspunkt (1996-1999)16,17,18,19,20,21 og mange andre strukturer er blevet offentliggjort til dato.
Her er hovedformålet at præsentere alternative protokoller til rensning af tryptofansynthansyntese og optimere proteinkrystallisering. Nærværende arbejde viser betydelige forbedringer for at rense den vilde type isoleret α-subunit (αStTS), isoleret β-subunit (βStTS), rekonstitueret α2β2StTS kompleks fra isolerede underenheder, og vilde type og mutant former af α2β2StTS kompleks. Fordelene i forhold til tidligere protokoller er betydelige, da rensningstiden blev reduceret betydeligt, og krystallisering og kryoprotektion blev optimeret. Mutant former for α2β2StTS kompleks manipuleret i dette arbejde har krystalliseret nær samme tilstand, der anvendes til den vilde type form. Men fin krystallisering optimering var nødvendig for at opnå store enkelt krystaller af tilstrækkelig kvalitet til struktur bestemmelse ved nær atomare opløsning. Til dato er der 134 tryptophan synthase krystal strukturer deponeret i Protein Data Bank (PDB), regnskab 101, 31 og 2 krystal strukturer, henholdsvis for bakterier, archaea og eukaryote. Pænt, 73 strukturer tilhører S. enterica serovar typhimurium og 5 krystal strukturer af α2β2StTS kompleks har opløsning grænser højere end 1,50 Angstroms. Ikke overraskende blev 4 ud af 5 udarbejdet i vores forskningsgruppe (PDB IDs: 5CGQ ved 1,18 Å, 4HT3 ved 1,30 Å, 4HPJ ved 1,45 Å, 6DZ4 ved 1,45 Å opløsning). De raffinerede krystalstrukturer af mutant form af α2β2StTS kompleks forventes at give ny indsigt i den mekanisme og roller, som essentielle aminosyre rester involveret i L-Tryptophan syntese.

<table>
	<tbody>
		<tr>
			<td>15 mL 10 kDa filter</td>
			<td>MilliporeSigma</td>
			<td>UFC901024</td>
			<td>centrifugal filter unit</td>
		</tr>
		<tr>
			<td>15 mL 100 kDa filter</td>
			<td>MilliporeSigma</td>
			<td>UFC910024</td>
			<td>centrifugal filter unit</td>
		</tr>
		<tr>
			<td>2 mL cryogenic vials</td>
			<td>Corning</td>
			<td>CLS430489</td>
			<td>Cryogenic vials</td>
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		<tr>
			<td>2 mL microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-141</td>
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			<td>Hampton Research</td>
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			<td>12-565-270</td>
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			<td>ammonium sulfate</td>
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			<td>BamHI</td>
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			<td>bicine</td>
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			<td>BP210-100</td>
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			<td>Cesium hydroxide</td>
			<td>Acros Organics</td>
			<td>AC213601000</td>
			<td>Chemical</td>
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		<tr>
			<td>Chloramphenicol</td>
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			<td>BP904-100</td>
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			<td>dimethyl sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>D1391</td>
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		<tr>
			<td>dithiothreitol</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
			<td>Chemical</td>
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		<tr>
			<td>DNA Polymerase</td>
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			<td>F530S</td>
			<td>HF polymerase</td>
		</tr>
		<tr>
			<td>dNTP Set</td>
			<td>Invitrogen</td>
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			<td>dNTPs set</td>
		</tr>
		<tr>
			<td>EcoRI</td>
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			<td>R0104S</td>
			<td>Restriction enzyme</td>
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		<tr>
			<td>His-Trap columns</td>
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			<td>IPTG</td>
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		<tr>
			<td>Luria Bertani agar</td>
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			<td>BP1425-2</td>
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		<tr>
			<td>NaCl</td>
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			<td>S271-500</td>
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		<tr>
			<td>NcoI</td>
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			<td>R0193S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Ni-NTA affinity beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>R90115</td>
			<td>Ni-NTA agarose beads</td>
		</tr>
		<tr>
			<td>PEG 8000</td>
			<td>Fisher Scientific</td>
			<td>BP233-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Fisher Scientific</td>
			<td>44-865-0</td>
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		<tr>
			<td>pyridoxal phosphate</td>
			<td>Acros Organics</td>
			<td>AC228170010</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>S-200 HR</td>
			<td>Cytiva</td>
			<td>45-000-196</td>
			<td>Size exclusion column</td>
		</tr>
		<tr>
			<td>SacI</td>
			<td>New England Biolabs</td>
			<td>R0156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sorvall RC-5B centrifuge</td>
			<td>Sorvall</td>
			<td>8327-30-1004</td>
			<td>Floor cetrifuge</td>
		</tr>
		<tr>
			<td>Spermine</td>
			<td>Acros Organics</td>
			<td>AC132750010</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Superdex 200 prep grade</td>
			<td>Cytiva</td>
			<td>45-002-491</td>
			<td>Size exclusion column</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td>DNA liagse</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Fisher Scientific</td>
			<td>BP152-500</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Ubl-specific protease 1</td>
			<td>Thermo Scientific</td>
			<td>12588018</td>
			<td>SUMO Protease</td>
		</tr>
	</tbody>
</table>

pcr mutagenesis, cloning, expression, fast protein purification protocols and crystallization of the wild type and mutant forms of tryptophan synthase

Strukturelle undersøgelser med tryptophan synthase (TS) bienzym kompleks (α2β2 TS) fra Salmonella typhimurium er blevet udført for bedre at forstå sin katalytiske mekanisme, allosterisk adfærd, og detaljer om den enzymatiske transformation af substrat til produkt i PLP-afhængige enzymer. I dette værk tillod et nyt udtrykssystem til fremstilling af de isolerede α- og isolerede β-underenhed rensning af store mængder rene underenheder og α2β2StTS-kompleks fra de isolerede underenheder inden for 2 dage. Rensningen blev udført ved affinitetskromatografi efterfulgt af kavalergang af affinitetsmærket, ammoniumsulfatudfældning og størrelsesudelukkelseskromatografi (SEC). For bedre at forstå den rolle, som nøglerester spiller ved enzymet β-site, blev site-direct mutagenesis udført i tidligere strukturelle undersøgelser. En anden protokol blev oprettet for at rense den vilde type og mutant α2β2StTS komplekser. En enkel, hurtig og effektiv protokol ved hjælp af ammoniumsulfatfraktionering og SEC tillod rensning af α2β2St TS-komplekspå en enkelt dag. Begge rensningsprotokoller, der er beskrevet i dette arbejde, har betydelige fordele sammenlignet med tidligere protokoller for at rense det samme kompleks ved hjælp af PEG 8000 og spermin til at krystalisere α2β2St TS-komplekslangs renseprotokollen. Krystallisering af vildtype og visse mutantformer forekommer under lidt forskellige forhold, der forringer rensningen af nogle mutanter ved hjælp af PEG 8000 og spermin. For at forberede krystaller egnet til røntgen krystallografiske undersøgelser blev der gjort en indsats for at optimere krystallisering, krystalkvalitet og kryoprotektion. De her præsenterede metoder bør generelt kunne anvendes til rensning af tryptofansynteseunderenheder og vilde typer og mutante α2β2St.TS-komplekser.

Rensning af α-og β- underenheder af tryptophan synthase Den α- underenhed (αStTS) og β-underenheden (βSt.TS) af Salmonella typhimurium tryptophan synthase blev subkloneret i den modificerede pET SUMO-vektor. Figur 1A viser repræsentative SDS-PAGE-resultater af to stærke bånd svarende til Fusionsproteinet His6-SUMO-αStTS (lane αON) o...

Watch this Scientific Journal Video about PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase at JoVE

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Denne artikel præsenterer en række på hinanden følgende metoder til udtryk og rensning af Salmonella typhimurium tryptophan synthase comp denne protokol et hurtigt system til at rense protein kompleks på en dag. Overdækkede metoder er stedsstyret mutagenese, proteinudtryk i Escherichia coli, affinitetskromatografi, gelfiltreringskromatografi og krystallisering.

PCR Mutagenesis, Kloning, Expression, Fast Protein Rensning Protokoller og krystallisering af wild type og mutant former for Tryptophan Synthase

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