Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158.
The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB. The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner. A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed. RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays. Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% alpha-helices and 20% beta-strands. Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 degrees C, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage.
Ruiz-Masó
José A
JA
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Velázquez, 144, E-28006 Madrid, Spain.
López-Zumel
Consuelo
C
Menéndez
Margarita
M
Espinosa
Manuel
M
del Solar
Gloria
G
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002942:Circular Dichroism; D004261:DNA Replication; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D012984:Software; D013696:Temperature; D014461:Ultracentrifugation / Q000379:methods
D001426:Bacterial Proteins; C048817:RepB protein, Bacteria