Solution and electron microscopy characterization of lactococcal phage baseplates expressed in Escherichia coli.
We report here the characterization of several large structural protein complexes forming the baseplates (or part of them) of Siphoviridae phages infecting Lactococcus lactis: TP901-1, Tuc2009 and p2. We revisited a "block cloning" expression strategy and extended this approach to genomic fragments encoding proteins whose interacting partners have not yet been clearly identified. Biophysical characterization of some of these complexes using circular dichroism and size exclusion chromatography, coupled with on-line light scattering and refractometry, demonstrated that the over-produced recombinant proteins interact with each other to form large (up to 1.9MDa) and stable baseplate assemblies. Some of these complexes were characterized by electron microscopy confirming their structural homogeneity as well as providing a picture of their overall molecular shapes and symmetry. Finally, using these results, we were able to highlight similarities and differences with the well characterized much larger baseplate of the myophage T4.
Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098 CNRS and Universités Aix-Marseille I & II, Campus de Luminy, Case 932, Marseille Cedex 09, France. valerie.campanacci@afmb.univ-mrs.fr
Valérie
V
Campanacci
David
D
Veesler
Julie
J
Lichière
Stéphanie
S
Blangy
Giuliano
G
Sciara
Sylvain
S
Moineau
Douwe
D
van Sinderen
Patrick
P
Bron
Christian
C
Cambillau
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D013294:Lactococcus lactis / Q000821:virology*; D008854:Microscopy, Electron; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D017906:Siphoviridae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012996:Solutions; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D012996:Solutions; D014764:Viral Proteins