Microbiology news and views
J. Mol. Micro. Biotechnol. 3: 83-93
OspA, a Lipoprotein Antigen of the Obligate Intracellular Bacterial Pathogen
Piscirickettsia salmonis
Michael A. Kuzyk, Ján Burian, Julian C. Thornton, and William W. Kay
No effective recombinant vaccines are currently available for any rickettsial diseases. In this regard the first non-ribosomal DNA sequences from the obligate
intracellular pathogen Piscirickettsia
salmonis are presented. Genomic DNA isolated from Percoll density gradient purified
P. salmonis, was used to construct an
expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal antiserum raised against
P. salmonis, with a bias toward P.
salmonis surface antigens, was used to identify immunoreactive clones. Catabolite repression of the
lac promoter was required to obtain a stable clone of a 4,983 bp insert in
Escherichia coli due to insert toxicity exerted by the accompanying
radA open reading frame (ORF). DNA sequence analysis of the insert revealed 1 partial and 4 intact
predicted ORF's. A 486 bp ORF, ospA, encoded a 17 kDa antigenic outer surface protein (OspA) with 62% amino acid sequence homology to the genus common 17 kDa
outer membrane lipoprotein of Rickettsia
prowazekii, previously thought confined to members of the genus
Rickettsia. Palmitate incorporation demonstrated that OspA
is posttranslationally lipidated in E. coli, albeit poorly expressed as a lipoprotein even after replacement of the signal sequence with the signal sequence from
lpp (Braun lipoprotein) or the rickettsial 17 kDa homologue. To enhance expression,
ospA was optimized for codon usage in E. coli
by PCR synthesis. Expression of ospA
was ultimately improved (~13% of total protein) with a truncated variant lacking a signal sequence. High level expression (~42% tot. prot.) was attained as an
N-terminal fusion protein with the fusion product recovered as inclusion bodies in
E. coli BL21. Expression of OspA in P.
salmonis was confirmed by immunoblot analysis
using polyclonal antibodies generated against a synthetic peptide of OspA (110-129) and a strong antibody response against OspA was detected in convalescent sera
from coho salmon (Oncorhynchus kisutch).
Full article [pdf]
New and forthcoming molecular biology books
Recommended Reading