Ringraziamo Stephan Wilkens aiuto in microscopia elettronica a trasmissione. Questo lavoro è stato sostenuto dal National Institutes of Health di Grant R01AG023731.

Precedenti studi hanno dimostrato che i primi amino-terminali 22 residui di Mgm101 sono spaccati dopo l&#39;importazione in mitocondri 19. Per l&#39;espressione in Escherichia coli, la cornice di lettura aperta MGM101 mancano i primi 22 codoni viene amplificato mediante PCR e collocato a valle della sequenza codificante la proteina MASCHIO legante il maltosio (MBP) in una versione modificata del vettore di espressione pMAL-c2e. Questo genera la fusione MBP-Mgm101 con un linker che contiene un sito di taglio per la proteasi prescissione (Figura 3). Il plasmide viene costruito selezionando E. trasformanti coli senza l&#39;IPTG e selezione blu / bianco Xgal. Il plasmide risultante pMAL-C2E-MGM101 viene poi introdotto nella E. coli ceppo BL21-CodonPlus (DE3)-RIL selezionando ampicillina e colonie resistenti cloramfenicolo. 1. Espressione, induzione, Cell Lysis e DNasi Trattamento io <ol><li> InocUlate trasformanti freschi in 10 ml di terreno LB (1% Bacto triptone, 0,5% estratto di lievito, 1% di NaCl) addizionato con glucosio (0,2%), ampicillina (100 pg / ml) e cloramfenicolo (50 mg / ml). Incubare a 37 ° CO / N con agitazione a 200 rpm. </li><li> Inoculare il ml precoltura 10 in 2 litri di terreno LB integrato come sopra. Crescere le cellule a 37 ° C con agitazione fino a OD 600 raggiunge 0,5. </li><li> Indurre l&#39;espressione della proteina di fusione MBP-Mgm101 aggiungendo isopropil β-D-1-tiogalattopiranoside (IPTG) ad una concentrazione finale di 0,3 mM. Crescere le cellule in agitazione a 30 ° C per 5 ore. </li><li> Raccogliere le cellule per centrifugazione utilizzando una Beckman JA-10 rotore (5.500 xg, a 4 ° C, 10 min). Dopo aver scartato il surnatante, risospendere il pellet cellulare in 30 ml di tampone di lisi (20 mM Tris-HCl, pH 7,4 e 1 mM EDTA, pH 7,4) contenente inibitori delle proteasi (25 pM leupeptina, 1 pM pepstatina A e 1 mM PMSF). </li><li> Sonicare sospensione cellulare in ghiaccio per 20sec utilizzando un processore ad ultrasuoni (Sistemi di calore; modello W-385) al duty cycle del 50%, lasciare raffreddare in ghiaccio per 30 secondi, e ripetere 2x. </li><li> Aggiungere 1 ml di DNAse 1 magazzino a 2 mg / ml. Rock il lisato cellulare a 4 ° C per 2 h. </li><li> Regolare NaCl ad una concentrazione finale di 500 mm. </li><li> Rimuovere i detriti cellulari mediante centrifugazione a 10.000 xg a 4 ° C per 30 min. </li></ol> 2. Purificazione con amilosio Affinity Chromatography <ol><li> Equilibrare 1,5 ml di resina amilosio (50% slurry) nel tampone di lisi. Aggiungere la resina amilosio equilibrata al lisato cellulare. Agitare delicatamente a 4 ° CO / N. </li><li> Caricare il mix lisato-resina su una colonna cromatografica Econo-colonna installata in una stanza fredda. Lasciare le proteine ​​non legate al passaggio della colonna per gravità. </li><li> Lavare la resina con amilosio 300 ml di tampone di lavaggio I (20 mM Tris-HCl, pH 7,4, 400 mM NaCl, 1 mM EDTA, pH 7,4, e 0,2 mM PMSF). </li><li> Ripetere il lavaggio con 150 ml di tampone di lavaggio II (20 mm tris-HCl, pH 7,4, 200 mM NaCl, 1 mM EDTA, pH 7,4, e 0,2 mM PMSF). </li><li> Aggiungere 5 ml di tampone di eluizione (20 mM Tris-HCl, pH 7,4, 200 mM NaCl, 1 mM EDTA, pH 7,4, e 0,2 mM PMSF, 10 mM maltosio) alla colonna, incubare a 4 ° C per 10 min, raccogliere l&#39;eluato e ripetere per più volte. </li><li> Combinare gli eluati, determinare la resa e la purezza di MBP-Mgm101 caricando una aliquota dell&#39;eluato su un gel SDS-PAGE seguita da colorazione con Coomassie (Figura 4). </li></ol> 3. Prescissione proteasi Decolleté e cromatografia a scambio cationico <ol><li> Aggiungere 50 unità di prescissione proteasi per l&#39;eluato MBP-Mgm101. Eseguire la scissione a 4 ° CO / N e lasciarlo continuare durante la dialisi contro il tampone di dialisi (20 mM Tris-HCl, pH 7,4, 100 mM NaCl, 2 mM DTT, 1 mM EDTA, pH 7,4, e 0,2 mM PMSF) </li><li> Controllare l&#39;efficienza della fenditura su un gel SDS-PAGE (Figura 5A). </li><li> Caricare il spaccati MBP-Mgm101 da iniettare multiplaioni su una cartuccia Mini Macro-Prep Alta S Bio-Scale collegati ad un sistema biologico FPLC doppio passaggio Bio-Rad. </li><li> Avviare la cromatografia a scambio cationico mediante l&#39;applicazione di un gradiente passo sale di 5 mm, 300 mm, 500 mm, 750 mm e 1000 mm di NaCl che viene creato da tampone di miscelazione A (5 mM NaCl, 10 mM Na-fosfato, pH 7,2; 1 mM PMSF) e tampone B (1 M NaCl, 10 mM Na-fosfato, pH 7,2, 1 mM PMSF). Impostare la velocità di flusso a 0,5 ml / min. Raccogliere la frazione Mgm101 e controllare la purezza della proteina su un gel SDS-PAGE (Figura 5B). </li></ol> 4. Size Exclusion Chromatography <ol><li> Unire le frazioni proteiche da cromatografia a scambio cationico. Dializzare la proteina in tampone di equilibrazione GF (20 mM MOPS, pH 7,0; 150 mM NaCl, 1 mM EDTA, pH 7,4, 5 mM di 2-mercaptoetanolo, 0,2 mM PMSF). </li><li> Concentrare la proteina con Vivaspin 15R colonnine ultrafiltrazione a ridurre il volume a ~ 1 ml mediante filatura a 3000 xga 4 ° C. </li><li> Caricare Mgm101 ona calibrato Superose 6 prep grade colonna equilibrata con il tampone di cromatografia (20 mM MOPS, pH 7,0; 150 mM NaCl, 5 mM β-mercaptoetanolo, 1 mM EDTA, pH 7,4; 0,2 mM PMSF). </li><li> Avviare la dimensione del buffer con esclusione cromatografia funzionare ad una velocità di flusso di 0,5 ml / min. </li><li> Raccogliere le frazioni di purificate Mgm101 picchi. Caricare aliquote delle frazioni su un 12% SDS-PAGE per il controllo della qualità finale della proteina. </li><li> Concentrato della proteina con Vivaspin 6, poi congelare rapidamente i campioni in N2 liquido e conservare a -80 ° C. </li><li> Utilizzare i campioni Mgm101 entro 6-12 mesi per i test ssDNA-binding (Figura 8) e per la visualizzazione strutturale di microscopia elettronica a trasmissione (Figura 9). </li></ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+dynamics+in+the+helical+filaments+formed+by+RecA+and+Rad51+on+DNA.">Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 8419-8424 (2001).</li><li>Conway, A. 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=Crystal+structure+of+a+Rad51+filament.">Crystal structure of a Rad51 filament.</a> Nat. Struct. Mol. Biol. 11, 791-796 (2004).</li><li>Bai, Y., Davis, A. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+allele+of+RAD52+that+causes+severe+DNA+repair+and+recombination+deficiencies+only+in+the+absence+of+RAD51+or+RAD59.">A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.</a> Genetics. 153, 1117-1130 (1999).</li><li>Bai, Y., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rad52+homolog+is+required+for+RAD51-independent+mitotic+recombination+in+Saccharomyces+cerevisiae.">A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.</a> Genes Dev. 10, 2025-2037 (1996).</li><li>Paques, F., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+of+recombination+induced+by+double-strand+breaks+in+Saccharomyces+cerevisiae.">Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.</a> Microbiol. Mol. Biol. Rev. 63, 349-404 (1999).</li><li>Lopes, A., Amarir-Bouhram, J., Faure, G., Petit, M. A., Guerois, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+novel+recombinases+in+bacteriophage+genomes+unveils+Rad52,+Rad51+and+Gp2.5+remote+homologs.">Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs.</a> Nucleic Acids Res. 38, 3952-3962 (2010).</li><li>Poteete, A. R., Sauer, R. T., Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+quaternary+organization+of+the+bacteriophage+P22+Erf+protein.">Domain structure and quaternary organization of the bacteriophage P22 Erf protein.</a> J. Mol. Biol. 171, 401-418 (1983).</li><li>Passy, S. I., Yu, X., Li, Z., Radding, C. M., Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rings+and+filaments+of+beta+protein+from+bacteriophage+lambda+suggest+a+superfamily+of+recombination+proteins.">Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 4279-4284 (1999).</li><li>Ploquin, M., 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=Functional+and+structural+basis+for+a+bacteriophage+homolog+of+human+RAD52.">Functional and structural basis for a bacteriophage homolog of human RAD52.</a> Curr. Biol. 18, 1142-1146 (2008).</li><li>Chen, X. J., Guan, M. X., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGM101,+a+nuclear+gene+involved+in+maintenance+of+the+mitochondrial+genome+in+Saccharomyces+cerevisiae.">MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae.</a> Nucl. Acids Res. 21, 3473-3477 (1993).</li><li>Meeusen, S., 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=Mgm101p+is+a+novel+component+of+the+mitochondrial+nucleoid+that+binds+DNA+and+is+required+for+the+repair+of+oxidatively+damaged+mitochondrial+DNA.">Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA.</a> J. Cell Biol. 145, 291-304 (1999).</li><li>Mbantenkhu, M., 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=Mgm101+is+a+Rad52-related+protein+required+for+mitochondrial+DNA+recombination.">Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.</a> J. Biol. Chem. 286, 42360-42370 (2011).</li><li>Nardozzi, J. D., Wang, X., Mbantenkhu, M., Wilkens, S., Chen, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+properly+configured+ring+structure+is+critical+for+the+function+of+the+mitochondrial+DNA+recombination+protein.">A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein.</a> Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).</li><li>Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+the+mitochondrial+genome+maintenance+protein+Mgm101p+in+Saccharomyces+cerevisiae+determined+with+a+temperature-conditional+allele.">A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.</a> FEMS Yeast Res. 7, 131-140 (2007).</li><li>Itoh, K., 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=DNA+packaging+proteins+Glom+and+Glom2+coordinately+organize+the+mitochondrial+nucleoid+of+Physarum+polycephalum.">DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum.</a> Mitochondrion. 11, 575-586 (2011).</li><li>Janicka, S., 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=A+RAD52-like+single-stranded+DNA+binding+protein+affects+mitochondrial+DNA+repair+by+recombination.">A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.</a> Plant J. 72, 423-435 (2012).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

E &#39;stata una sfida per la produzione di una stalla, nativo Mgm101 proteina ricombinante da E. coli forse a causa della sua insolubilità in cellule batteriche. In questo rapporto, si dimostra che la strategia di MBP-fusione permette la proteina da esprimere ad un livello ragionevolmente elevato. Mediante microscopia elettronica a colorazione negativa e cromatografia ad esclusione dimensionale, abbiamo precedentemente dimostrato che la proteina di fusione MBP-forma oligomeri uniformi in vitro 1...

Ricombinazione omologa è importante per la riparazione delle rotture del doppio filamento (DSB) e interstrand legami crociati, e per la ripresa della replicazione del DNA da forche di replicazione crollate 1. Nel convenzionale ricombinazione omologa, la reazione è catalizzata centrale dalle ricombinasi ATP-dipendenti comprese RecA nei procarioti e Rad51 e Dmc1 negli eucarioti 1-3. Questi ricombinasi formano filamenti nucleoproteici sul ssDNA, che sono essenziali per l&#39;avvio ricerca di omologia e invasione filone all&#39;interno dei modelli di DNA duplex (Figura 1, pannello di sinistra) 4-7. Oltre alla schema convenzionale, ricombinazione omologa può avvenire anche in maniera RecA/Rad51-independent (figura 1, pannello di destra). Per esempio, il lievito e RAD52 Rad59 proteine ​​possono catalizzare direttamente la ricottura del complementari ssDNA filamenti che sono esposti per resezione di dsDNA pause. Questo processo di ricombinazione, noto come cantareLe filone ricottura, generalmente non comporta omologa l&#39;abbinamento con i modelli di DNA a doppia elica. Dopo la ricottura, code eterologhe vengono rimossi da esonuleasi e nick sono legatura a ripristinare genoma 8-10 di continuità. Riparazione da parte del singolo meccanismo di ricottura filo è spesso accompagnata da delezioni di sequenze genomiche tra regioni ripetute direttamente. RAD52 appartiene ad un gruppo eterogeneo di proteine ​​di ricombinazione che sono diffuse tra i batteriofagi 11. Queste proteine ​​sono conosciute anche come singolo filamento Proteine ​​ricottura (SSAPs), in base alla loro attività nel promuovere la ricottura di molecole di DNA a singolo filamento omologo. I migliori SSAPs batteriofagi sono caratterizzati Redβ e Erf dal batteriofagi λ e P22, rect dal profago RAC e la proteina Sak dal Lactococcal fago ul36. Le SSAPs sono strutturalmente caratterizzate da una tipica piega β-β-β-α, sebbene somiglianza è praticamente undetectgrado nelle loro sequenze primarie. Essi ogni forma di grandi anelli omo-oligomeri di 10-14 volte simmetria in vitro 12-14. Le implicazioni funzionali di questa caratteristica organizzazione strutturale ordine superiore non è ben compreso. Siamo interessati a capire il meccanismo di ricombinazione omologa nel genoma mitocondriale. Abbiamo precedentemente identificato il gene MGM101 che è essenziale per il mantenimento del mtDNA in Saccharomyces cerevisiae 15. MGM101 stato successivamente trovato per essere associato con nucleoidi mitocondriali ed è necessario per la tolleranza del mtDNA ad agenti che danneggiano il DNA 16. Tuttavia, lo studio di Mgm101 è stata frenata nell&#39;ultimo decennio dalla difficoltà di produrre Mgm101 ricombinante. Abbiamo recentemente riusciti a produrre Mgm101 solubile in grandi quantità da E. coli usando la strategia di MBP-fusion. Questo ci ha permesso di dimostrare che Mgm101 azionisimilitudini biochimiche e strutturali con il RAD52-famiglia di proteine ​​17,18. In questo rapporto, una procedura di purificazione in tre fasi è descritta, che produce Mgm101for analisi biochimiche e strutturali omogenee (Figura 2).

<table><tbody><tr><td>Expression vector pMAL-c2E</td><td>New England Biolabs</td><td>#N8066S</td><td></td></tr><tr><td>PreScission Protease</td><td>GE Healthcare Life Sciences</td><td>#27-0843-01</td><td></td></tr><tr><td>BL21-CodonPlus(DE3)-RIL cells</td><td>Strategene</td><td>#230245</td><td></td></tr><tr><td>Leupeptin</td><td>Roche Applied Science</td><td>#11034626001</td><td></td></tr><tr><td>Pepstatin</td><td>Roche Applied Science</td><td>#11359053001</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Roche Applied Science</td><td>#10837091001</td><td></td></tr><tr><td>DNAse I</td><td>Sigma</td><td>#DN25-1G</td><td></td></tr><tr><td>Isopropyl &amp;#946;-D-1-thiogalactopyranoside (IPTG)</td><td>Roche Applied Science</td><td>#11411446001</td><td></td></tr><tr><td>Amylose resin</td><td>New England Biolabs</td><td>#E8021L</td><td></td></tr><tr><td>Econo-Column chromatography column</td><td>BIO-RAD</td><td>#7372512</td><td></td></tr><tr><td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td><td>BIO-RAD</td><td>#732-4130</td><td></td></tr><tr><td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS15RH02</td><td></td></tr><tr><td>Superose 6 prep grade column</td><td>Amersham Bioscirnces</td><td>#17-0489-01</td><td></td></tr><tr><td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td><td>Sartorius Stedium</td><td>#VS0611</td><td></td></tr></tbody></table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

Il gene è stato identificato MGM101 20 anni fa per il suo ruolo nel mantenimento del DNA mitocondriale. Studi condotti in diversi gruppi hanno suggerito che la proteina Mgm101 è coinvolta nella riparazione ricombinazione del DNA mitocondriale. Recenti indagini hanno indicato che Mgm101 è legato alla famiglia RAD52-tipo ricombinazione proteica. Queste proteine ​​formano grandi anelli oligomeriche e promuovere la ricottura di molecole di DNA a singolo filamento omologo. Tuttavia, la caratterizzazione di Mgm101 è stata ostacolata dalla difficoltà di produrre la proteina ricombinante. Qui, una procedura affidabile per la preparazione di Mgm101 ricombinante è descritto. Il maltosio Binding Protein (MBP)-tag Mgm101 è prima espressa in Escherichia coli. La proteina di fusione è inizialmente purificato mediante cromatografia di affinità amilosio. Dopo essere stato rilasciato da clivaggio proteolitico, Mgm101 è separato da MBP mediante cromatografia di scambio cationico. Monodisperso Mgm101 è quindi ottenutoper cromatografia ad esclusione dimensionale. Una resa di ~ 0,87 mg di Mgm101 per litro di coltura batterica può essere ottenuta routine. Il Mgm101 ricombinante ha la contaminazione minima di DNA. I campioni preparati sono utilizzati con successo per biochimici, strutturali e singola particella analisi dell&#39;immagine di Mgm101. Questo protocollo può essere utilizzato anche per la preparazione di altri grandi oligomeriche proteine ​​che legano il DNA che possono essere mal ripiegate e tossici per le cellule batteriche.

Mgm101 è una proteina di ricombinazione RAD52-correlati nei mitocondri. RAD52 è stato ampiamente studiato per il suo ruolo nella ricombinazione del DNA mitocondriale (Figura 1). Ricombinante Mgm101 può essere preparato da una procedura in tre fasi (Figura 2). Ciò è facilitato dall&#39;utilizzo della strategia MBP-tagging che permette Mgm101 essere espressi in una forma solubile e successivamente rilasciato dal tag da taglio proteolitico (Figura 3). <p class="jo...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Il lievito mitocondriale proteina nucleoide, Mgm101, è una proteina di ricombinazione RAD52-tipo che forma grandi anelli oligomeriche. Un protocollo è descritto da preparare Mgm101 solubile ricombinante utilizzando la proteina legante il maltosio (MBP)-tagging strategia accoppiato con scambio cationico e cromatografia ad esclusione dimensionale.

Preparazione del Mgm101 ricombinazione delle proteine ​​da Strategy Tagging MBP-based

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

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12.0K Views.  State University of New York Upstate Medical University. Il lievito mitocondriale proteina nucleoide, Mgm101, è una proteina di ricombinazione RAD52-tipo che forma grandi anelli oligomeriche. Un protocollo è descritto da preparare Mgm101 solubile ricombinante utilizzando la proteina legante il maltosio (MBP)-tagging strategia accoppiato con scambio cationico e cromatografia ad esclusione dimensionale.

Video: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Ingegneria fattori artificiali per manipolare in modo specifico l&#39;alternanza di splicing nelle cellule umane

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Ricerca

Genetica

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Inducibile T7 RNA Polymerase-mediato sistema di espressione multigene, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

Integrazione mirata CRISPR/Cas9-mediata In Vivo usando una strategia basata su unendo fine omologia-mediata

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

La manipolazione genetica in<em&gt; Δku80</em&gt; Ceppi per analisi genomica funzionale di<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Immunologia e infezioni

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish Modello di Metastasi del Neuroblastoma

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Ricerca sul cancro

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Fotoattivato localizzazione Microscopia con complementazione fluorescenza biomolecolare (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Bioingegneria

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

Arricchimento di vescicole extracellulari native e ricombinanti di micobatteri

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subcloning più Insertion (SPI) - A Novel Metodo recombineering per la costruzione rapida di gene targeting Vettori

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Biologia

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.

One-step CRISPR-based Strategy for Endogenous Gene Tagging in Drosophila melanogaster

Here, we present a simplified endogenous gene tagging protocol for Drosophila, which utilizes a PCR-based technique for marker-free identification of successful genetic modifications, facilitating the development of stable knock-in lines.

Strategia basata su CRISPR in un'unica fase per il tagging genico endogeno in Drosophila melanogaster

The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that ...

Neuroscienze

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Preparazione del Mgm101 ricombinazione delle proteine da Strategy Tagging MBP-based

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ingegneria fattori artificiali per manipolare in modo specifico l'alternanza di splicing nelle cellule umane

Inducibile T7 RNA Polymerase-mediato sistema di espressione multigene, pMGX

Integrazione mirata CRISPR/Cas9-mediata In Vivo usando una strategia basata su unendo fine omologia-mediata

La manipolazione genetica in

Zebrafish Modello di Metastasi del Neuroblastoma

Fotoattivato localizzazione Microscopia con complementazione fluorescenza biomolecolare (BiFC-PALM)

Arricchimento di vescicole extracellulari native e ricombinanti di micobatteri

Subcloning più Insertion (SPI) - A Novel Metodo recombineering per la costruzione rapida di gene targeting Vettori

Strategia basata su CRISPR in un'unica fase per il tagging genico endogeno in Drosophila melanogaster

Modificazione genetica dei cianobatteri mediante congiugazione utilizzando il toolkit di clonazione modulare CyanoGate

Preparazione del Mgm101 ricombinazione delle proteine ​​da Strategy Tagging MBP-based

Riepilogo

Esplora Altri Video

Capitoli in questo video

Ingegneria fattori artificiali per manipolare in modo specifico l'alternanza di splicing nelle cellule umane

Inducibile T7 RNA Polymerase-mediato sistema di espressione multigene, pMGX

Integrazione mirata CRISPR/Cas9-mediata In Vivo usando una strategia basata su unendo fine omologia-mediata

La manipolazione genetica in

Zebrafish Modello di Metastasi del Neuroblastoma

Fotoattivato localizzazione Microscopia con complementazione fluorescenza biomolecolare (BiFC-PALM)

Arricchimento di vescicole extracellulari native e ricombinanti di micobatteri

Subcloning più Insertion (SPI) - A Novel Metodo recombineering per la costruzione rapida di gene targeting Vettori

Strategia basata su CRISPR in un'unica fase per il tagging genico endogeno in Drosophila melanogaster

Modificazione genetica dei cianobatteri mediante congiugazione utilizzando il toolkit di clonazione modulare CyanoGate

Preparazione del Mgm101 ricombinazione delle proteine da Strategy Tagging MBP-based