Vi erkänner Dr Amir Aharoni för hans stöd och Geula Davidov, Noam Grimberg och Chen Guttman för deras råd och kommentarer.

1. Kloning och uttryck mama Gene i E. coli Den muterade genen mamAΔ41 förstärktes med polymeraskedjereaktion (PCR) från genomiska DNA Magnetospirillum magneticum AMB-1, med grundfärg: 5&#39;-GCATTACGCATATGGACGACATCCGCCAGGTG-3 &#39;och 5&#39;-GCGCGGCAGCCATA-TGGCATACG-3 &quot;. I den förstärkta DNA-fragment, var en NcoI plats infördes i början kodon ATG och uppsägning kodon ersattes med en ScoI webbplats. Fragmenten var kokas med NcoI och SACI och kl...

<ol>
	<li>Faivre, D., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+Bacteria+and+Magnetosomes.">Magnetotactic Bacteria and Magnetosomes.</a> Chem Rev. 108, 4875-4898 (2008).</li><li>D'Andrea, L. D., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TPR+proteins:+the+versatile+helix.">TPR proteins: the versatile helix.</a> Trends Biochem Sci. 28, 655-662 (2003).</li><li>Young, J. C., Barral, J. M., Hartl, U. l. r. i. c. h., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+folding:+localized+functions+of+cytosolic+chaperones.">More than folding: localized functions of cytosolic chaperones.</a> Trends Biochem Sci. 28, 541-547 (2003).</li><li>Brocard, C., Hartig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+targeting+signal+1:+is+it+really+a+simple+tripeptide?.">Peroxisome targeting signal 1: is it really a simple tripeptide?.</a> Biochim Biophys Acta. 1763, 1565-1573 (2006).</li><li>Fransen, M., Amery, L., Hartig, A., Brees, C., Rabijns, A., Mannaerts, G. P., Van Veldhoven, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+PTS1-+and+Rab8b-binding+properties+of+Pex5p+and+Pex5Rp/TRIP8b.">Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b.</a> Biochim Biophys Acta. 1783, 864-873 (2008).</li><li>Baker, M. J., Frazier, A. E., Gulbis, J. M., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein-import+machinery:+correlating+structure+with+function.">Mitochondrial protein-import machinery: correlating structure with function.</a> Trends Cell Biol. 17, 456-464 (2007).</li><li>Mirus, O., Bionda, T., von Haeseler, A., Schleiff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionarily+evolved+discriminators+in+the+3-TPR+domain+of+the+Toc64+family+involved+in+protein+translocation+at+the+outer+membrane+of+chloroplasts+and+mitochondria.">Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria.</a> J Mol Model. 15, 971-982 (2009).</li><li>Gatsos, X., Perry, A. J., Anwari, K., Dolezal, P., Wolynec, P. P., Likic, V. A., Purcell, A. W., Buchanan, S. K., Lithgow, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+and+outer+membrane+assembly+in+Alphaproteobacteria.">Protein secretion and outer membrane assembly in Alphaproteobacteria.</a> FEMS Microbiol Rev. 32, 995-1009 (2008).</li><li>Tiwari, D., Singh, R. K., Goswami, K., Verma, S. K., Prakash, B., Nandicoori, 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=Key+residues+in+Mycobacterium+tuberculosis+protein+kinase+G+play+a+role+in+regulating+kinase+activity+and+survival+in+the+host.">Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.</a> J Biol Chem. 284, 27467-27479 (2009).</li><li>Edqvist, P. J., Broms, J. E., Betts, H. J., Forsberg, A., Pallen, M. J., Francis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetratricopeptide+repeats+in+the+type+III+secretion+chaperone,+LcrH:+their+role+in+substrate+binding+and+secretion.">Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.</a> Mol Microbiol. 59, 31-44 (2006).</li><li>Grunberg, K., Muller, E. C., Otto, A., Reszka, R., Linder, D., Kube, M., Reinhardt, R., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+proteomic+analysis+of+the+magnetosome+membrane+in+Magnetospirillum+gryphiswaldense.">Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense.</a> Appl Environ Microbiol. 70, 1040-1050 (2004).</li><li>Komeili, A., Vali, H., Beveridge, T. J., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosome+vesicles+are+present+before+magnetite+formation,+and+MamA+is+required+for+their+activation.">Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation.</a> Proc Natl Acad Sci USA. 101, 3839-3843 (2004).</li><li>Okuda, Y., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+a+magnetosome-associated+protein,+TPR-containing+MAM22,+in+Escherichia+coli.">Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli.</a> FEBS Lett. 491, 169-173 (2001).</li><li>Taoka, A., Asada, R., Sasaki, H., Anzawa, K., Wu, L. F., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localizations+of+Mam22+and+Mam12+in+the+magnetosomes+of+Magnetospirillum+magnetotacticum.">Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum.</a> J Bacteriol. 188, 3805-3812 (2006).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41, 207-234 (2005).</li></ol>

Protein rening är det viktigaste steget i alla proteiner biokemiska eller strukturella studier. Eftersom varje protein är unik med sitt eget beteende måste man definiera dess egenskaper och ändra dess rening därefter. Protein mål bör analyseras som ett första steg mot rening med hjälp av bioinformatiska verktyg. De används för att beräkna målet iso-elektriska punkten, bedöma behovet av att minska / oxiderande miljön och dess behov av speciella joner / ligander. Det finns flera viktiga ändringar av våra ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French Press</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-078A</td>
<td>&nbsp;</td>
<tr>
<td>Pressure cell</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-032</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge</td>
<td>Equipment</td>
<td>Sorvall</td>
<td>Discovery 90SE</td>
<td>&nbsp;</td>
<tr>
<td>Rottor</td>
<td>Equipment</td>
<td>Beckman</td>
<td>Ti60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml</td>
<td>Equipment</td>
<td>Beckman</td>
<td>BC-355618</td>
<td>&nbsp;</td>
<tr>
<td>2.5cm diameter, Glass Econo-Column Chromatography Columns</td>
<td>Equipment</td>
<td>BioRad</td>
<td>737-2521</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA His Bind resin</td>
<td>Equipment</td>
<td>Novagen</td>
<td>M0063428</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer</td>
<td>Equipment</td>
<td>Amersham Biosiences</td>
<td>Ultraspec 2100 pro</td>
<td>&nbsp;</td>
<tr>
<td>Quartz cuvette</td>
<td>Equipment</td>
<td>Hellma</td>
<td>104-QS</td>
<td>&nbsp;</td>
<tr>
<td>Fast Performance Liquid Chromatography- AKTA purifier 10</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>28-4062-64</td>
<td>&nbsp;</td>
<tr>
<td>Ion exchange column &#x2013; MonoQ 4.6/100 PE</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>10025543</td>
<td>&nbsp;</td>
<tr>
<td>Size exclusion pre-packed column-HiLoad 26/60 Superdex 200</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>17-1071-01</td>
<td>&nbsp;</td>
<tr>
<td>Centricon - Vivaspin15 &#x2013; 10,000 MWCO</td>
<td>Equipment</td>
<td>Sartorius Stedim Biotech GmbH</td>
<td>VS1501</td>
<td>&nbsp;</td>
<tr>
<td>Table centrifuge</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>IEC CL30R</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>Equipment</td>
<td>Bruker Daltonics</td>
<td>Reflex IV</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl (hydrotymethyl) aminomethane</td>
<td>Reagent</td>
<td>BioLab</td>
<td>20092391</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>FRUTROM</td>
<td>235553470</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole</td>
<td>Reagent</td>
<td>Alfa Aesar</td>
<td>288-32-4</td>
<td>&nbsp;</td>
<tr>
<td>EDTA free protease inhibitors cocktail</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-8849</td>
<td>&nbsp;</td>
<tr>
<td>Dnase I (Deoxyribonuclease I)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>DN-25</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Thrombin</td>
<td>Reagent</td>
<td>Fisher BioReagents</td>
<td>BP25432</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>BioLab</td>
<td>07132391</td>
<td>&nbsp;</td>
<tr>
<td>Soudim Dodecyl Sulfate (SDS)</td>
<td>Reagent</td>
<td>BioLab</td>
<td>19822391</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-3148</td>
<td>&nbsp;</td>
<tr>
<td>InstantBlue</td>
<td>Reagent</td>
<td>Expedeon</td>
<td>1SB01L</td>
<td>&nbsp;</td>
<tr>
<td>PageRuler Prestained Protein Ladder</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>SM0671</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification of the m. magneticum strain amb-1 magnetosome associated protein mama&delta;41

Magnetotactic bakterier utgör en mångskiftande grupp vattenlevande mikroorganismer som har möjlighet att orientera sig längs geomagnetiska fält. Detta beteende tros Stödet sitt sökande efter lämpliga miljöer<em&gt; (1)</em&gt;. Denna funktion är knutna till magnetosome, blåsor en subcellulära organell som består av en linjär kedja montering av lipid varje möjlighet att biomineralize och bifoga en ~ 50-nm kristaller av magnetit eller greigite. En princip komponent i magnetosome som visat sig krävas för att bilda funktionella blåsor är mamma. Mamma är en mycket riklig magnetosome-associerad protein som är en av de mest kännetecknas magnetosome-associerade proteiner<em&gt; In vivo</em&gt;<em&gt; (2-6)</em&gt;. Denna artikel fokuserar på rening av mamma, som trots studeras<em&gt; In vivo</em&gt; Har ingen tydlig funktionell eller strukturell information identifierats för det. Bioinformatik analys föreslog att mamma är en tetra-tricopeptide upprepa (TPR) som innehåller protein. TPR är en strukturell motiv finns som sådan eller som ingår i en större vik i ett brett spektrum av proteiner, fungerar den som en mall för protein-protein interaktioner och förmedlar flera proteinkomplex<em&gt; (7)</em&gt;. TPRs är inblandade i många viktiga uppgifter i eukaryota celler organell processer och många bakteriella vägar<em&gt; (8-14).</em&gt; För att förstå mamma, en unik TPR innehåller protein är renat protein krävs som ett första steg. I denna artikel presenterar vi reningen protokollet för en stabil Mama radering mutant (MamAΔ41) från<em&gt; M. magneticum</em&gt; AMB-1.

Watch this Scientific Journal Video about Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÃŽâ€41 at JoVE.com

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÃƒÅ½Ã¢â‚¬Â41

Mamma är en unik Magnetosome tillhörande protein som visade sig vara involverad i magnetosome aktivering. Här presenterar vi rening protokollet för Mama radering mutant (MamAΔ41) från<em&gt; M. magneticum</em&gt; AMB-1.

Rening av M. magneticum Sila AMB-1 Magnetosome Medverkande Protein MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Research

JoVE Journal

Biology

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Färgning av proteinerna i geler med Coomassie G-250 utan organiska lösningsmedel och ättiksyra

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Använda GELFREE 8100 Fraktionering System för Molekylär viktbaserad fraktionering med vätskefasen Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Purification of Progenitors from Skeletal Muscle

Rening av stamceller från skelettmuskulatur

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Automatiserad Hydrofob Interaktion kromatografikolonn val för användning i Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Purification of Hsp104, a Protein Disaggregase

Rening av Hsp104, ett protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Identifiering av Protein Samverkande Partners Använda Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Expression och rening av den cystisk fibros transmembran konduktans-regulator protein i Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Spirande jästprotein extraktion och rening för studien av funktion, interaktioner, och post-translationella modifieringar

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Identifiering av posttranslationella modifieringar av växtproteinkomplex

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Rening av cystisk fibros transmembran konduktansregulator Protein Uttryckt i<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...