In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90°C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80°C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80°C. Interestingly, addition of the dye to the denatured VHH-R2 at 80°C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibodyantigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications. Proteins 2005;59:555-564.
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.
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