Recombinant Yersinia enterocolitica YscM1 and YscM2: homodimer formation and susceptibility to thrombin cleavage.
Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) make use of a virulence plasmid-encoded type three secretion system (TTSS) to inject effector proteins into host cells. Y. enterocolitica YscM1 (LcrQ in Y. pestis and Y. pseudotuberculosis) and its homologue YscM2 are regulatory components of the TTSS that are also secreted by this transport apparatus. YscM1 and YscM2 share 57% identity and are believed to be functionally equivalent. We have recombinantly expressed and purified YscM1 and YscM2 in Escherichia coli. After expression as glutathione S-transferase (GST) fusions purification to near homogeneity was achieved by glutathione-Sepharose affinity chromatography followed by PreScission protease treatment to cleave off GST and gel filtration on a Superdex 75 column. Such recombinant YscM1 and YscM2 bound efficiently to the specific chaperone SycH, indicating proper folding of the purified proteins. Gel filtration analyses revealed that both YscM1 and YscM2 formed homodimers. The YscM1 and YscM2 homodimers could be dissociated at high ionic strength, indicating that salt bridges essentially contribute to the dimerization. We further demonstrated that YscM1 and YscM2 are susceptible to thrombin cleavage.
Wilharm
Gottfried
G
Department of Bacteriology, Max von Pettenkofer-Institut, Pettenkoferstr 9a, Munich D-80336, Germany. maier@m3401.mpk.med.uni-muenchen.de
Neumayer
Wibke
W
Heesemann
Jürgen
J
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014157:Transcription Factors; C110346:YscM1 protein, Yersinia enterocolitica; C110345:YscM2 protein, Yersinia enterocolitica; D013917:Thrombin