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

Abstract

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.

Cyclic Di-AMP Homeostasis in Bacillus subtilis

“…Briefly, growing cultures of B. subtilis were treated with formaldehyde (0.6%, w/v; 20 min) to facilitate cross-linking of interacting proteins (24). The Strep-tagged proteins and their potential inter- action partners were then purified from crude extracts using a StrepTactin column (IBA, Göttingen, Germany) and desthiobiotin as the eluent.…”

“…For the fusion of the Strep tag to the C terminus of the target protein, we constructed plasmid pGP1460 essentially in the same way. However, the fragment covering the promoter of the degQ Hy gene, the multiple cloning site, and DNA coding for the Strep tag was obtained with pGP382 (24) and the primer pair M13-fwd/HE308.…”

“…Detection of Protein-Protein Interactions-The isolation of protein complexes from B. subtilis cells was performed using Strep-protein interaction experiment technology (24). Briefly, growing cultures of B. subtilis were treated with formaldehyde (0.6%, w/v; 20 min) to facilitate cross-linking of interacting proteins (24).…”

“…The promoter-Strep tag fragment was obtained by PCR using plasmid pGP380 (24) and the primer pair M13-fwd/HE307 and subsequent digestion with EcoRI and BglII. The resulting plasmid was pGP1459.…”

Current knowledge on regulatory RNAs and their machineries inStaphylococcus aureus

“…Although there is no homolog of RNase E in Gram-positive bacteria, 109 several enzymes were proposed to be organized into a multi-enzymatic complex in S. aureus 26 and B. subtilis. 110,111 Using various strategies, networks of interactions have been identified between two glycolytic enzymes (enolase and phosphofructokinase), the DEAD-box RNA helicase CshA and four RNases, namely RNase J1, RNase J2, RNase Y and PNPase. 26,110,112 The two B. subtilis enzymes RNase J1/J2 are endowed with a dual activity of an endoribonuclease and a 5'-3' exoribonuclease 113 while RNase Y, is the functional equivalent of RNase E and cleaves mRNA at unpaired U/A rich sequences.…”