Difference between revisions of "SPINE"

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(Created page with 'SPINE is a methhod to detect in vivo protein-protein interactions A detailed protocol to detect the interaction between RocG and GltC: 1 litre of a ''Bacillus subtilis'' cult...')
 
(→‎Studies that made use of SPINE:)
 
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SPINE is a methhod to detect in vivo protein-protein interactions  
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'''SPINE is a method to detect ''in vivo'' protein-protein interactions''' [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+17994626 PubMed]
  
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A detailed protocol to detect the interaction between RocG and GltC:
 
  
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1 litre of a ''Bacillus subtilis'' culture was grown to an OD600 of approx. 1.0 and incubated with 0.6% formaldehyde (stock solution 4% in PBS, pH 6.5!) for 20 minutes @ 37°C on a shaker.  
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==[http://www.iba-lifesciences.com/IBA-Applications-Protein-interaction-SPINE-Technology.html '''See the principle''' ]==
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=='''A detailed protocol to detect the interaction between [[RocG]] and [[GltC]]:''' ==
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1 litre of a ''B. subtilis'' culture was grown to an OD<sub>600</sub> of approx. 1.0 and incubated with 0.6% formaldehyde ( 4% stock solution in PBS, pH 6.5!) for 20 minutes @ 37°C on a shaker.  
 
The cells were harvested and washed once in 1 X PBS pH 6.5.  
 
The cells were harvested and washed once in 1 X PBS pH 6.5.  
 
The pellets can then be stored @ -20 °C.
 
The pellets can then be stored @ -20 °C.
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The GltC protein was expressed carrying a Strep-tag and RocG expression was induced by arginine (see Commichau et al., 2007 Mol Microbiol). Expression of the Strep-tagged GltC protein allows to test the functionality of the protein.  
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The [[GltC]] protein was expressed carrying a Strep-tag and [[RocG]] expression was induced by arginine ([http://www.ncbi.nlm.nih.gov/sites/entrez/17608797 PubMed]). Expression of the Strep-tagged GltC protein allows to test the functionality of the protein.  
 
Crude extracts (10-15 ml) were prepared by using a French Press.
 
Crude extracts (10-15 ml) were prepared by using a French Press.
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After a centrifugation step for 1 h @ 27.000 g the clarified crude extracts were loaded onto a Streptactin sepharose column (0.5-1 ml matrix) to isolate the cross-linked protein complexes (the detailed procedure for protein purification is described in the IBA manual, http://www.iba-go.com/).
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After a centrifugation step for 1 h @ 27.000 g the clarified crude extracts were loaded onto a Streptactin sepharose column (0.5-1 ml matrix) to isolate the cross-linked protein complexes (the detailed procedure for protein purification is described in the IBA manual, http://www.iba-go.com/).
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After the purification of the protein complexes the crosslinks can be resolved by boiling the samples in laemmli buffer for 10-15 minutes @ 95 °C (Herzberg et al., 2007 Proteomics).
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After the purification of the protein complexes the crosslinks can be resolved by boiling the samples in Laemmli buffer for 10-15 minutes @ 95 °C ([http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+17994626 PubMed]).
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A 12.5% SDS gel was loaded with the samples and the proteins were then visualized by silver-staining. We identified the interaction partner/s by mass spectroscopy and Western blotting.  
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A 12.5% SDS gel was loaded with the samples and the proteins were then visualized by silver-staining. The interaction partner/s were identified by mass spectroscopy and Western blotting.  
  
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Preparation of the formaldehyde stock solution (max. 4% in 1X PBS pH 6.5):
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Preparation of the formaldehyde stock solution (max. 4% in 1 X PBS pH 6.5):
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We use paraformaldehyde (a white powder). Paraformaldehyde can be solved in 1 X PBS for approx. 20-30 minutes @ 65 to 70 °C.  
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We use ''para''-formaldehyde (a white powder; http://en.wikipedia.org/wiki/Paraformaldehyde). ''para''-formaldehyde dissolves within approx. 20-30 minutes in 1 X PBS for @ 65 to 70 °C.  
  
 
The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/).
 
The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/).
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=='''Relevant plasmids:'''==
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for use in ''B. subtilis'' (multicopy plasmids): [[pGP380]], [[pGP382]]
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for use in ''B. subtilis'' (chromosomal integration under the control of the native promoter): [[pGP1389]]
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for use in ''E. coli'': [[pGP172]], [[pGP574]]
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=='''Biotin-containing proteins that are purified with the Strep-Tactin column'''==
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[[PycA]], [[AccB]]
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=='''The reference for the method:'''==
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<pubmed> 17994626 </pubmed>
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=='''SPINE for membrane proteins:'''==
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<pubmed> 21472855 24300168 </pubmed>
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=='''Studies that made use of SPINE:'''==
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<pubmed> 17608797  19193632 20572937 20933603 21622759 21803996 21992469 22001508 22517742 23192352 22512862 24325460 24375102 24666271 25711804 </pubmed>
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=='''The use of SPINE in other microbes:'''==
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<pubmed> 21123179 </pubmed>

Latest revision as of 09:18, 9 March 2015

SPINE is a method to detect in vivo protein-protein interactions PubMed


See the principle

A detailed protocol to detect the interaction between RocG and GltC:

1 litre of a B. subtilis culture was grown to an OD600 of approx. 1.0 and incubated with 0.6% formaldehyde ( 4% stock solution in PBS, pH 6.5!) for 20 minutes @ 37°C on a shaker. The cells were harvested and washed once in 1 X PBS pH 6.5. The pellets can then be stored @ -20 °C. The GltC protein was expressed carrying a Strep-tag and RocG expression was induced by arginine (PubMed). Expression of the Strep-tagged GltC protein allows to test the functionality of the protein. Crude extracts (10-15 ml) were prepared by using a French Press. After a centrifugation step for 1 h @ 27.000 g the clarified crude extracts were loaded onto a Streptactin sepharose column (0.5-1 ml matrix) to isolate the cross-linked protein complexes (the detailed procedure for protein purification is described in the IBA manual, http://www.iba-go.com/). After the purification of the protein complexes the crosslinks can be resolved by boiling the samples in Laemmli buffer for 10-15 minutes @ 95 °C (PubMed). A 12.5% SDS gel was loaded with the samples and the proteins were then visualized by silver-staining. The interaction partner/s were identified by mass spectroscopy and Western blotting.

Preparation of the formaldehyde stock solution (max. 4% in 1 X PBS pH 6.5): We use para-formaldehyde (a white powder; http://en.wikipedia.org/wiki/Paraformaldehyde). para-formaldehyde dissolves within approx. 20-30 minutes in 1 X PBS for @ 65 to 70 °C.

The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/).

Relevant plasmids:

for use in B. subtilis (multicopy plasmids): pGP380, pGP382

for use in B. subtilis (chromosomal integration under the control of the native promoter): pGP1389

for use in E. coli: pGP172, pGP574

Biotin-containing proteins that are purified with the Strep-Tactin column

PycA, AccB

The reference for the method:

Christina Herzberg, Lope Andrés Flórez Weidinger, Bastian Dörrbecker, Sebastian Hübner, Jörg Stülke, Fabian M Commichau
SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo.
Proteomics: 2007, 7(22);4032-5
[PubMed:17994626] [WorldCat.org] [DOI] (P p)


SPINE for membrane proteins:

Volker Steffen Müller, Karolin Tschauner, Sabine Hunke
Membrane-SPINE: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo.
J Vis Exp: 2013, (81);e50810
[PubMed:24300168] [WorldCat.org] [DOI] (I e)

Volker S Müller, Peter R Jungblut, Thomas F Meyer, Sabine Hunke
Membrane-SPINE: an improved method to identify protein-protein interaction partners of membrane proteins in vivo.
Proteomics: 2011, 11(10);2124-8
[PubMed:21472855] [WorldCat.org] [DOI] (I p)


Studies that made use of SPINE:

Lorena Stannek, Martin J Thiele, Till Ischebeck, Katrin Gunka, Elke Hammer, Uwe Völker, Fabian M Commichau
Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis.
Environ Microbiol: 2015, 17(9);3379-90
[PubMed:25711804] [WorldCat.org] [DOI] (I p)

Julia Domínguez-Escobar, Diana Wolf, Georg Fritz, Carolin Höfler, Roland Wedlich-Söldner, Thorsten Mascher
Subcellular localization, interactions and dynamics of the phage-shock protein-like Lia response in Bacillus subtilis.
Mol Microbiol: 2014, 92(4);716-32
[PubMed:24666271] [WorldCat.org] [DOI] (I p)

Sabrina Graf, Dominik Schmieden, Karolin Tschauner, Sabine Hunke, Gottfried Unden
The sensor kinase DctS forms a tripartite sensor unit with DctB and DctA for sensing C4-dicarboxylates in Bacillus subtilis.
J Bacteriol: 2014, 196(5);1084-93
[PubMed:24375102] [WorldCat.org] [DOI] (I p)

Maike Bartholomae, Frederik M Meyer, Fabian M Commichau, Andreas Burkovski, Wolfgang Hillen, Gerald Seidel
Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites.
FEBS J: 2014, 281(4);1132-43
[PubMed:24325460] [WorldCat.org] [DOI] (I p)

Felix M P Mehne, Katrin Gunka, Hinnerk Eilers, Christina Herzberg, Volkhard Kaever, Jörg Stülke
Cyclic di-AMP homeostasis in bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth.
J Biol Chem: 2013, 288(3);2004-17
[PubMed:23192352] [WorldCat.org] [DOI] (I p)

Alexander K W Elsholz, Kürsad Turgay, Stephan Michalik, Bernd Hessling, Katrin Gronau, Dan Oertel, Ulrike Mäder, Jörg Bernhardt, Dörte Becher, Michael Hecker, Ulf Gerth
Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis.
Proc Natl Acad Sci U S A: 2012, 109(19);7451-6
[PubMed:22517742] [WorldCat.org] [DOI] (I p)

Andrea Wünsche, Elke Hammer, Maike Bartholomae, Uwe Völker, Andreas Burkovski, Gerald Seidel, Wolfgang Hillen
CcpA forms complexes with CodY and RpoA in Bacillus subtilis.
FEBS J: 2012, 279(12);2201-14
[PubMed:22512862] [WorldCat.org] [DOI] (I p)

Frederik M Meyer, Matthieu Jules, Felix M P Mehne, Dominique Le Coq, Jens J Landmann, Boris Görke, Stéphane Aymerich, Jörg Stülke
Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.
J Bacteriol: 2011, 193(24);6939-49
[PubMed:22001508] [WorldCat.org] [DOI] (I p)

Jens J Landmann, Ricarda A Busse, Jan-Hendrik Latz, Kalpana D Singh, Jörg Stülke, Boris Görke
Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis in Bacillus subtilis.
Mol Microbiol: 2011, 82(3);770-87
[PubMed:21992469] [WorldCat.org] [DOI] (I p)

Martin Lehnik-Habrink, Joseph Newman, Fabian M Rothe, Alexandra S Solovyova, Cecilia Rodrigues, Christina Herzberg, Fabian M Commichau, Richard J Lewis, Jörg Stülke
RNase Y in Bacillus subtilis: a Natively disordered protein that is the functional equivalent of RNase E from Escherichia coli.
J Bacteriol: 2011, 193(19);5431-41
[PubMed:21803996] [WorldCat.org] [DOI] (I p)

A K W Elsholz, K Hempel, S Michalik, K Gronau, D Becher, M Hecker, U Gerth
Activity control of the ClpC adaptor McsB in Bacillus subtilis.
J Bacteriol: 2011, 193(15);3887-93
[PubMed:21622759] [WorldCat.org] [DOI] (I p)

Frederik M Meyer, Jan Gerwig, Elke Hammer, Christina Herzberg, Fabian M Commichau, Uwe Völker, Jörg Stülke
Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon.
Metab Eng: 2011, 13(1);18-27
[PubMed:20933603] [WorldCat.org] [DOI] (I p)

Martin Lehnik-Habrink, Henrike Pförtner, Leonie Rempeters, Nico Pietack, Christina Herzberg, Jörg Stülke
The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex.
Mol Microbiol: 2010, 77(4);958-71
[PubMed:20572937] [WorldCat.org] [DOI] (I p)

Fabian M Commichau, Fabian M Rothe, Christina Herzberg, Eva Wagner, Daniel Hellwig, Martin Lehnik-Habrink, Elke Hammer, Uwe Völker, Jörg Stülke
Novel activities of glycolytic enzymes in Bacillus subtilis: interactions with essential proteins involved in mRNA processing.
Mol Cell Proteomics: 2009, 8(6);1350-60
[PubMed:19193632] [WorldCat.org] [DOI] (I p)

Fabian M Commichau, Christina Herzberg, Philipp Tripal, Oliver Valerius, Jörg Stülke
A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC.
Mol Microbiol: 2007, 65(3);642-54
[PubMed:17608797] [WorldCat.org] [DOI] (P p)


The use of SPINE in other microbes:

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