Name: identification of plant ice-binding proteins through assessment of ice-recrystallization inhibition and isolation using ice-affinity purification
Uploaded: 2017-05-05T13:00:00
Description: Watch this Scientific Journal Video about Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification at JoVE.com

Dette arbejde blev finansieret af en NSERC (Canada) tilskud til VKW.

1. Splat Apparat Setup <ol><li> Fyld en temperatur programmerbar cirkulerende vandbad med ethylenglycol (50% volumen / volumen i vand). BEMÆRK: Grøn automotive ethylenglycol kan anvendes. </li><li> For at samle det eksterne kammer, anvender isolerede plastrør til at fastgøre vandbad til en dobbeltvægget glasskål. Isolere glasskål med polystyrenskum og tildækkes med en plastpetriskål låg. Skær en 3-4 tommer hul i bunden af ​​polystyren kammeret, således at lyskilden kan ses gennem glasset kammer....

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+antifreeze+protein+in+a+fish.">Hyperactive antifreeze protein in a fish.</a> Nature. 429 (6988), 153 (2004).</li><li>Zhang, D. Q., Liu, B., Feng, D. R., He, Y. M., Wang, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification+and+antifreeze+activity+of+carrot+antifreeze+protein+and+its+mutants.">Expression, purification and antifreeze activity of carrot antifreeze protein and its mutants.</a> Protein Expr. Purif. 35 (2), 257-263 (2007).</li><li>Gupta, R., Dreswal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+temperature+stress+modulated+secretome+analysis+and+purification+of+antifreeze+protein+from+Hippophae+rhamnoides,+a+Himalayan+wonder+plant.">Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant.</a> Proteome Res. 11 (5), 2684-2696 (2012).</li><li>Kuiper, M. J., Lankin, C., Gauthier, S. Y., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+antifreeze+proteins+by+adsorption+to+ice.">Purification of antifreeze proteins by adsorption to ice.</a> Biochem. Biphys. Res. Commun. 300 (3), 645-648 (2003).</li><li>Middleton, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+ice-binding+proteins+from+higher+plants.">Isolation and characterization of ice-binding proteins from higher plants.</a> Methods Mol. Biol. 1166, 255-277 (2014).</li><li>Bredow, M., Vanderbeld, B., Walker, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-binding+proteins+confer+freezing+tolerance+in+transgenic+Arabidopsis+thaliana.">Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana.</a> Plant Biotechnol. J. , (2016).</li><li>Olijve, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+rapid+ice+crystal+growth+through+nonbasal+plane+adsorption+of+antifreeze+proteins.">Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 113 (14), 3740-3745 (2016).</li><li>Zakharov, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+Recrystallization+in+a+Solution+of+a+Cryoprotector+and+Its+inhibition+by+a+Protein:+Synchrotron+X-Ray+Diffraction+Study.">Ice Recrystallization in a Solution of a Cryoprotector and Its inhibition by a Protein: Synchrotron X-Ray Diffraction Study.</a> J. Pharm. Sci. 105 (7), 2129-2138 (2016).</li><li>Capicciotti, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+ice+recrystallization+inhibitors+enable+freezing+of+human+red+blood+cells+with+reduced+glycerol+concentrations.">Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations.</a> Sci. Rep. 5, 9692 (2015).</li><li>Knight, C. A., Cheng, C. C., DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+alpha-helical+antifreeze+peptides+on+specific+ice+crystal+surface+planes.">Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes.</a> Biophys. 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For vellykket analyse og oprensning af IBPS, er det vigtigt at forstå den temperaturfølsomme karakter af nogle af disse proteiner. Bestemt plante IBPS bliver ustabile ved temperaturer over 0 ° C, hvilket resulterer i udfoldning, udfældning og inaktivitet. For at opnå aktive IBPS, er det ofte kritisk, at planterne behandles i et koldt rum (~ 4 ° C) og Prøverne opbevares på is under eksperimentering. En anden faktor at overveje ved brug helcelle rålysater er nedbrydningen af ​​proteiner ved endogene proteaser...

Is-bindende proteiner (IBPS) er en forskelligartet familie af beskyttende proteiner der er blevet opdaget i en række organismer, herunder planter 1, insekter 2, fisk 3, og mikrober 4. Det centrale element i disse proteiner er deres enestående evne til specifikt og effektivt adsorbere til iskrystaller, ændre deres vækst. IBPS har flere dokumenterede egenskaber, med de to mest velkarakteriserede være termisk hysterese (TH) og is-omkrystallisation inhibering (IRI). TH aktivitet lettere observeret i IBPS produceret i freeze-intolerant dyr. Denne aktivitet resulterer i sænkning af frysepunktet for organismernes kredsløbs- eller interstitielle væsker for at forhindre frysning. I modsætning hertil i fryse-tolerante organismer, som uundgåeligt vil fryse ved temperaturer under frysepunktet, IBPS synes at have lav TH aktivitet. Trods den lave TH aktivitet, til en høj IRI aktivitet begrænse iskrysstal vækst er ofte observeret med disse proteiner. For fryse-tolerante organisme, denne IRI aktivitet formentlig hjælper med at beskytte celler fra den ukontrollerede vækst af is i ekstracellulære rum. Den &quot;madras knappen&quot; model kan anvendes til at beskrive den mekanisme hvorved IBPS forhindre vækst af iskrystaller 5. Under denne model, IBPS specifikt adsorbere til iskrystallen overflade med mellemrum, således at vandmolekyler kun kan optage med den voksende is krystalgitteret i mellemrummet mellem bundet IBPS. Dette skaber en krumning, der gør inkorporering af yderligere vandmolekyler ugunstige, en begivenhed, der kan beskrives ved den Gibbs-Thomson-effekten 6. Det forankrede clathrathydrater farvande hypotese tilvejebringer en mekanisme til den specifikke binding af IBPS til iskrystallen overflade hvorved tilstedeværelsen af ​​ladede rester specifikt placeret på proteinet is bindingsstedet, resulterer i reorganization af vandmolekyler, så de matcher et eller flere planer af isen krystalgitteret 7. TH-aktivitet kan kvantificeres ved at måle forskellen mellem smelte- og frostgrader af en enkelt iskrystal i nærvær af en IBP. Mens TH aktivitet er en bredt accepteret fremgangsmåde til evaluering af aktiviteten af ​​IBPS, den lave TH kampen produceret af planten IBPS (typisk kun en brøkdel af en grad) kræver normalt en høj proteinkoncentration, specialudstyr og operatør erfaring. Selvom ikke-IBPS kan begrænse iskrystalvækst, er det en egenskab deles af alle IBPS og dermed testning for IRI aktivitet er en effektiv indledende screening for tilstedeværelsen af ​​IBPS, især for dem med lav TH-aktivitet. Den anvendte metode til at teste denne virkning er kendt som en &#39;splat assay&#39;, hvorved et protein prøve lynfrosset til frembringelse af et monolag af små iskrystaller, som er observeret over en tidsperiode for at bestemme, omiskrystalvækst er begrænset. I modsætning til andre metoder, der anvendes til at screene en kilde vævsprøve for tilstedeværelsen af ​​IBPS, denne teknik kan anvendes på lave koncentrationer protein i området 10-100 ng, anvender let-fabrikeret udstyr, og genererer data, der er hurtigt og nemt tolkes. Det er imidlertid vigtigt at understrege, at dette assay tilvejebringer en indledende screening for IBPS der bør følges ved bestemmelse af TH og iskrystal formning. Isolering af native proteiner er udfordrende, hvilket ofte kræver information vedrørende de strukturelle og biokemiske egenskaber af et protein af interesse. Affiniteten af ​​IBPS til ice muliggør isoleringen af ​​disse proteiner ved anvendelse is som et substrat til oprensningsformål. Da hovedparten af ​​molekyler skubbes fremad for grænsen mellem isvand under iskrystalvækst, langsom vækst af et is-halvkugle i nærvær af en IBP prøven resulterer i en højt oprenset prøve, blottet for stor quantiterne af kontaminerende proteiner og opløste stoffer. Denne fremgangsmåde er blevet anvendt til at identificere IBPS fra insekter 8, 9, 10, bakterier 11, fisk 12 og planter 13, 14. Desuden kan IBP-berigede fraktioner opnået gennem denne metode også anvendes til nedstrøms biokemisk analyse. Dette papir beskriver identifikationen af ​​IBPS i planter gennem induktion og udvinding af IBPS, at analyse af IRI aktivitet bekræfte tilstedeværelsen af ​​proteiner, og isoleringen af ​​proteiner under anvendelse af is affinitetsoprensning.

<table border="0" cellpadding="0" cellspacing="0" width="1117">
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL microcentrifugetubes</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">05-408-129</td>
			<td style="width:631px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adjustable lab jack</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">S63080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge</td>
			<td style="width:152px;">Desaga MC2</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brass probe</td>
			<td style="width:152px;">Custom built</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera/ camera port</td>
			<td style="width:152px;">Canon</td>
			<td style="width:119px;"></td>
			<td>Canon Power Shot SX110 digitial camera; custom built microscope port</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cheesecloth</td>
			<td style="width:152px;">Purewipe/Fisher Scientific</td>
			<td style="width:119px;">06-665-25A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentration tubes (0.5 mL)</td>
			<td style="width:152px;">EMD Millipore</td>
			<td style="width:119px;">UFC501008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentration tubes (15 mL)</td>
			<td style="width:152px;">EMD Millipore</td>
			<td style="width:119px;">UFC900308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tubes (50 mL)</td>
			<td style="width:152px;">Thermo Fisher</td>
			<td style="width:119px;">AM12502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cooling block</td>
			<td style="width:152px;">VWR</td>
			<td style="width:119px;">13259</td>
			<td>Use a metal heating block</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dehumidifier</td>
			<td style="width:152px;">Whirlpool</td>
			<td style="width:119px;"></td>
			<td>50 pint Energy Star dehumidifier; purchase from local supplier</td>
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			<td height="21" style="height:21px;width:216px;">Dessciation beads</td>
			<td style="width:152px;">t.h.e. Dessicant/VWR</td>
			<td style="width:119px;">EM-DX0017-2</td>
			<td>6-8 mesh size; 100% indicating</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:152px;">Olympus Tokyo</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double walled glass bowl</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Purchase from local lab glassware supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dry ice</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Use local supplier; hazardous&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA-protease inhibitor tablets</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td>11836170001</td>
			<td>Roche cOmplete mini</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Green automotive ethylene glycol can be purchased from any local hardware store (i.e. Home Depot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexane</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">296090</td>
			<td>Anhydrous, 95%; hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Immersion oil</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">56822</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">JA10/20 centrifuge</td>
			<td style="width:152px;">Beckman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large plastic petri dish</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid nitrogen</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Use local supplier; hazardous&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stir plate</td>
			<td style="width:152px;">Hanna Instruments</td>
			<td style="width:119px;">HI190M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope cover slides</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic tube</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Purchase PVC pipe from local hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polarized film</td>
			<td style="width:152px;">Edmund Optics</td>
			<td style="width:119px;">43-781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polystyrene foam</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td>Can be constructed from polystyrene shipping boxes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poreclain mortar and pestle</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">FB961</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVPP</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:119px;">77627</td>
			<td>110 &#181;m particle size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retort Stand</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">12-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small stir bar</td>
			<td style="width:152px;">Fisher</td>
			<td style="width:119px;">14-513-51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-programmable water bath</td>
			<td style="width:152px;">VWR</td>
			<td style="width:119px;">13271-118</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vacuum grease</td>
			<td style="width:152px;">Dow Corning/Sigma Aldrich</td>
			<td style="width:119px;">Z273554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vinyl tubing</td>
			<td style="width:152px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of plant ice-binding proteins through assessment of ice-recrystallization inhibition and isolation using ice-affinity purification

Is-bindende proteiner (IBPS) tilhører en familie af stressinducerede proteiner, der syntetiseres af visse organismer, der udsættes for frost. I planter, frostskader opstår, når ekstracellulære iskrystaller vokser, hvilket resulterer i brud af plasmamembraner og eventuel celledød. Adsorption af IBPS til iskrystaller begrænser yderligere vækst ved en proces kendt som is-omkrystallisation inhibering (IRI), og derved reducere cellulær skade. IBPS demonstrerer også evne til at sænke frysepunktet af en opløsning under ligevægtstemperaturen smeltepunktet, en egenskab kendt som termisk hysterese (TH) aktivitet. Disse beskyttende egenskaber har rejst interesse i identifikationen af ​​hidtil ukendte IBPS grund af deres potentielle anvendelse i industrielle, medicinske og landbrugsmæssige anvendelser. Dette papir beskriver identifikation af plante IBPS gennem 1) induktionen og udvinding af IBPS i plantevæv, 2) screening af ekstrakter til IRI aktivitet, og 3) isolering og purifIcation af IBPs. Efter induktion af IBP&#39;er ved lav temperatur eksponering testes ekstrakter for IRI aktivitet ved anvendelse af en &#39;splat-analyse&#39;, som muliggør observation af iskrystalvækst ved anvendelse af et standardlysmikroskop. Dette assay kræver en lavproteinkoncentration og frembringer resultater, som hurtigt opnås og fortolkes let, hvilket tilvejebringer en indledende skærm for isbindende aktivitet. IBP&#39;er kan derefter isoleres fra forurenende proteiner ved at udnytte IBP&#39;ernes egenskaber til at adsorbere til is ved hjælp af en teknik kaldet &#39;isaffinitetsrensning&#39;. Ved anvendelse af cellelysater opsamlet fra planteekstrakter kan en ishalvkugle langsomt dyrkes på en messingprobe. Dette inkorporerer IBP&#39;er i den krystallinske struktur af den polykrystallinske is. Kræver ingen biokemisk eller strukturel viden om IBP&#39;en, giver denne metode mulighed for genopretning af aktivt protein. Isrensede proteinfraktioner kan anvendes til downstream-applikationer, herunder identifikation af peptidevand sekvenser ved massespektrometri og den biokemiske analyse af native proteiner.

For at lette opsætning, figur 1 og Figur 2 er inkluderet som visuelle repræsentationer af udstyr til IRI analyse og is-affinitetsoprensning hhv. Resultaterne af IRI analyse under anvendelse af ekstrakter opsamlet fra sennep ukrudt med og uden IBPS er afbildet i figur 3. Disse resultater viser, at ekstrakter indsamlet fra sennep ukrudt, som ikke er fryse-tolerant, var ude af stan...

Watch this Scientific Journal Video about Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification at JoVE.com

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Dette papir beskriver identifikation af is-bindende proteiner fra freeze-tolerante planter gennem vurderingen af ​​is-omkrystallisation inhiberingsaktivitet og efterfølgende isolering af native IBPS anvender is-affinitetsoprensning.

Identifikation af Plant Ice-bindende Proteiner Gennem Vurdering af Ice-omkrystallisation inhibering og isolering ved hjælp af is-affinitet Oprensning

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

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Biochemistry

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Opsamling og identifikation af RNA-bindende proteiner ved hjælp af klik kemi-assisteret RNA-interactome Capture (CARIC) strategi

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

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The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

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