SPINE

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

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 1X PBS pH 6.5): We use para-formaldehyde (a white powder). 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: pGP380, pGP382

for use in E. coli: pGP172, pGP574

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

PycA, AccB

Other studies that made use of SPINE

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)