Construction of a Chimeric Shuttle Plasmid via a Heterodimer System: Secretion of an scFv Protein from Bacillus brevis Cells Capable of Inhibiting Hemagglutination

Functional Analysis of the Thioredoxin Domain inPorphyromonas gingivalisHBP35

“…This approach allowed the following: once a set of almost identical plasmids with only a difference in the drugresistance genes was prepared, it was easy to exchange certain fragments, which is flanked by unique restriction sites, from one plasmid to another. 13) We believe that unless these two unique techniques are combined, T4 DNA polymerase-mediated rapid introduction of mutagenesis and the heterodimer system, it might be impossible to accomplish such studies.…”

“…This approach allowed us to exchange certain fragments, flanked by unique restriction sites, from one plasmid to another, 13) as shown in Fig. 3.…”

“…Therefore, this plasmid was referred to as a heterodimer plasmid. 13) The hybrid plasmid was digested with restriction enzyme MfeI, ligated, and transformed into E. coli JM109. By selecting the transformants on agar plates containing Ap, an E. coli strain harboring plasmid Ap 3-Ser (D) was obtained.…”

“…To introduce site-directed mutagenesis, we exploited a simple, rapid, and inexpensive approach based on the utilization of only the T4 DNA polymerase, and all four codons coding for Cys residues present in the hbp35 were successfully altered to ones coding for Ser residues. In addition, the mutation points created were combined by a novel technique, designated the heterodimer system, 13) and plasmids with double and multiple mutations were also constructed.…”

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

“…brevis strains have been successfully used as a host for the production of recombinant proteins with biotechnological or pharmaceutical interest such as cytokines, hormones and single chain antibodies, based on multicopy plasmid expression systems able to promote secretion of encoded proteins into the growth media (Kozuka et al 1996, Udaka et al 1989, Udaka and Yamagata 1993, Shiroya et al 2001, Takimura et al 1997. Of particular interest for the vaccine field was the production of recombinant toxin subunits of B. pertussis and the LTB subunit of LT produced by ETEC strains, to be used either as antigens for subunit vaccines or as a mucosal adjuvant in the case of LTB, by recombinant B. brevis strains (Byun et al 2001, Kozuka et al 2000.…”