Yersinia enterocolitica type III secretion chaperone SycD: recombinant expression, purification and characterization of a homodimer.
Yersinia species pathogenic to human benefit from a protein transport machinery, a type three secretion system (T3SS), which enables the bacteria to inject effector proteins into host cells. Several of the transport substrates of the Yersinia T3SS, called Yops (Yersinia outer proteins), are assisted by specific chaperones (Syc for specific Yop chaperone) prior to transport. Yersinia enterocolitica SycD (LcrH in Yersinia pestis and Yersinia pseudotuberculosis) is a chaperone dedicated to the assistance of the translocator proteins YopB and YopD, which are assumed to form a pore in the host cell membrane. In an attempt to make SycD amenable to structural investigations we recombinantly expressed SycD with a hexahistidine tag in Escherichia coli. Combining immobilized nickel affinity chromatography and gel filtration we obtained purified SycD with an exceptional yield of 120mg per liter of culture and homogeneity above 95%. Analytical gel filtration and cross-linking experiments revealed the formation of homodimers in solution. Secondary structure analysis based on circular dichroism suggests that SycD is mainly composed of alpha-helical elements. To prove functionality of purified SycD previously suggested interactions of SycD with Yop secretion protein M2 (YscM2), and low calcium response protein V (LcrV), respectively, were reinvestigated.
Schmid
Annika
A
Max von Pettenkofer-Institut, Lehrstuhl für Bakteriologie, Ludwig-Maximilians-Universität München, Pettenkoferstr. 9a, D-80336 München, Germany.
Dittmann
Svea
S
Grimminger
Valerie
V
Walter
Stefan
S
Heesemann
Jürgen
J
Wilharm
Gottfried
G
D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D052899:Pore Forming Cytotoxic Proteins; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015008:Yersinia enterocolitica / Q000737:chemistry / Q000235:genetics
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C043908:LcrV protein, Yersinia; D018832:Molecular Chaperones; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; C118339:SycD protein, bacteria; D014157:Transcription Factors; C110345:YscM2 protein, Yersinia enterocolitica