Cloning, E. coli overexpression, purification and binding properties of TraA and TraC, two proteins involved in the pheromone-dependent conjugation process in enterococci.
The bacteriocin encoding plasmid pPD1 from Enterococcus faecalis is involved in a mating response to the sex pheromone cPD1 produced by recipient bacterial cells devoid of pPD1. Previous studies showed that cPD1 is internalized into donor cells in a process in which TraC plays the role of cell surface pheromone receptor. Inside the recipient cells, the pheromone binds to the plasmid-encoded cytoplasmic protein TraA, able to recognize specific DNA sequences and to modulate the conjugation process. To avoid self-induction of the conjugation process, donor cells produce the inhibitor iPD1, which competes with cPD1. This study was designed to produce recombinant TraA and TraC in a functionally active state and to evaluate their main functional properties. We have isolated the sequences encoding TraA and TraC from the plasmid pPD1 and cloned them in suitable expression vectors. The two recombinant proteins were successfully obtained in a soluble form using Escherichia coli as expression host and a T7 inducible expression system. TraC and TraA were purified to homogeneity by three or two chromatographic steps, respectively, leading to a final yield up to 4mg/l of cell culture for TraC and up to 10mg/l of cell culture for TraA. The ability of TraA and TraC to bind the specific pheromone and inhibitor peptides has been assessed by means of ESI-mass spectrometry. Moreover, the ability of recombinant TraA to bind DNA has been demonstrated by means of electrophoretic mobility shift assay. Overall these results are consistent with the heterologously expressed TraC and TraA being functionally active.
Alfieri
Beatrice
B
Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Viale Usberti 23/A, 43100 Parma, Italy.
Folloni
Silvia
S
Elviri
Lisa
L
Gobbo
Marina
M
Berni
Rodolfo
R
Folli
Claudia
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D013293:Enterococcus faecalis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010675:Pheromones / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D017931:DNA Primers; D029968:Escherichia coli Proteins; D010675:Pheromones; D011994:Recombinant Proteins