Proteomic analysis and identification of the structural and regulatory proteins of the Rhodobacter capsulatus gene transfer agent.
The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf's 3, 5, 6-9, 11, 13, and 15.
Chen
Frank
F
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Spano
Anthony
A
Goodman
Benjamin E
BE
Blasier
Kiev R
KR
Sabat
Agnes
A
Jeffery
Erin
E
Norris
Andrew
A
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Lebedev
Nikolai
N
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D022761:Gene Transfer, Horizontal; D008969:Molecular Sequence Data; D005810:Multigene Family; D046912:Multiprotein Complexes / Q000737:chemistry / Q000648:ultrastructure; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016354:Rhodobacter capsulatus / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D010455:Peptides; D020543:Proteome; D011993:Recombinant Fusion Proteins