Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.
Immunoconjugates have been widely studied as potential therapeutics for infectious diseases to direct unspecific antimicrobials to pathogens. In this study, the recombinant approach was used for expression of the immunoconjugate composed of the variable domain of a llama heavy-chain antibody (VHH) against Streptococcus mutans and dhvar5, a synthetic antimicrobial peptide. Before cloning, the impact of the elongation of the peptide termini on its biological activity was evaluated by chemical synthesis of the N- or C-termini extended dhvar5 peptides. As the elongation of the C-terminus had a greater influence on decline of the antimicrobial activity, the N-terminal fusion was designed. To promote in vivo release of the active peptide, a factor Xa cleavage site was inserted between VHH and dhvar5. Propagation of transformed Escherichia coli with the constructed plasmid was only possible in the absence of isopropyl beta-D-thiogalactoside (IPTG). Although these data demonstrate that the diminished antimicrobial activity of dhvar5 by the N-terminal fusion to VHH was not sufficient for the protection of the bacterial host cells against the peptide lethal effect, an insight into propeptides biological activities may be beneficial not only for new and more successful rearrangement of the VHH-dhvar5 immunoconjugate construct, but also design of the other recombinant molecules composed of peptides toxic to host cells.
Monoclonal antibodies play an important role in the development of diagnostic assays. Instead of using hybridoma technology to isolate human immunodeficiency virus type 1-specific antibodies, a phagedisplayed antibody library was generated from a small number (10 7 ) of peripheral blood lymphocytes from a seropositive donor. Two families of singlechain antibodies (scFvs) were selected by biopanning with the envelope precursor gp160. ELISA and competition in the BlAcore system revealed that one antibody family recognized a conformationsensitive epitope within gp120, while the other antibody family was gp41-specific. The latter group had sequence similarity to antibodies recognizing
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