SPINE: A method for the rapid detection and analysis of protein–protein interactions in vivo

The detection and analysis of protein–protein interactions is one of the central tasks of proteomics in the postgenomic era. For this purpose, we present a procedure, the Strep–protein interaction experiment (SPINE) that combines the advantages of the Strep‐tag protein purification system with those of reversible in vivo protein crosslinking by formaldehyde. Using two Bacillus subtilis regulator proteins, we demonstrate that this method is well suited to isolate protein complexes with high purity and virtually no background. Plasmids allowing the high‐level expression of proteins carrying an N‐ or C‐terminal Strep‐tag in B. subtilis were constructed.

[1]  K. Terpe.  Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems , 2002, Applied Microbiology and Biotechnology.

[2]  James R. Knight,et al.  A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae , 2000, Nature.

[3]  Jörg Stülke,et al.  A regulatory protein–protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC , 2007, Molecular microbiology.

[4]  D. Zühlke,et al.  A tyrosine kinase and its activator control the activity of the CtsR heat shock repressor in B. subtilis , 2005, The EMBO journal.

[5]  W. Hillen,et al.  Mycoplasma pneumoniae HPr kinase/phosphorylase. , 2004, European journal of biochemistry.

[6]  Abraham L. Sonenshein,et al.  Modulation of Activity of Bacillus subtilis Regulatory Proteins GltC and TnrA by Glutamate Dehydrogenase , 2004, Journal of bacteriology.

[7]  S. Ishikawa,et al.  A new FtsZ‐interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis , 2006, Molecular microbiology.

[8]  Fabian M. Commichau,et al.  Characterization of Bacillus subtilis Mutants with Carbon Source-Independent Glutamate Biosynthesis , 2006, Journal of Molecular Microbiology and Biotechnology.

[9]  B. Séraphin,et al.  A generic protein purification method for protein complex characterization and proteome exploration , 1999, Nature Biotechnology.

[10]  R. Ozawa,et al.  A comprehensive two-hybrid analysis to explore the yeast protein interactome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Wojcik,et al.  The protein–protein interaction map of Helicobacter pylori , 2001, Nature.

[12]  A. Sonenshein,et al.  Mechanism of repression by Bacillus subtilis CcpC, a LysR family regulator. , 2003, Journal of molecular biology.

[13]  W. Hillen,et al.  Protein kinase‐dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram‐positive bacteria , 1995, Molecular microbiology.

[14]  G. Rapoport,et al.  The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon , 1995, Journal of bacteriology.

[15]  J. Stülke,et al.  Regulation of citB expression in Bacillus subtilis: integration of multiple metabolic signals in the citrate pool and by the general nitrogen regulatory system , 2006, Archives of Microbiology.

[16]  M. Débarbouillé,et al.  Interactions of wild-type and truncated LevR of Bacillus subtilis with the upstream activating sequence of the levanase operon. , 1994, Journal of molecular biology.

[17]  A. Galinier,et al.  Interaction of GapA with HPr and Its Homologue, Crh: Novel Levels of Regulation of a Key Step of Glycolysis in Bacillus subtilis? , 2006, Journal of bacteriology.

[18]  S. Ehrlich,et al.  Actin homolog MreBH governs cell morphogenesis by localization of the cell wall hydrolase LytE. , 2006, Developmental cell.

[19]  Juergen Kast,et al.  Identification of protein‐protein interactions using in vivo cross‐linking and mass spectrometry , 2004, Proteomics.

[20]  P. Uetz,et al.  The elusive yeast interactome , 2006, Genome Biology.

[21]  J. Errington,et al.  Multiple Interactions between the Transmembrane Division Proteins of Bacillus subtilis and the Role of FtsL Instability in Divisome Assembly , 2006, Journal of bacteriology.