Lactobacillus helveticus S-layer protein specific binders from antibody libraries

Many strains of Lactobacillus helveticus have been characterised as probiotics. These strains were reported to exert health benefits such as protection against infection, e.g. by modulating the immune system. L. helveticus MIMLh5, isolated from Grana Padano cheese natural whey starter, was proposed as a potential probiotic bacterium for the human pharynx with promising antagonistic and immunomodulatory properties (Guglielmetti et al., 2010). The surface layer (S-layer) protein was identified as being involved in the immunomodulatory properties of L. helveticus strains M92 (Beganović et al., 2011), MIMLh5 (Guglielmetti and Taverniti, unpublished data). Antibodies constitute a powerful tool to study protein function, protein localisation and protein-protein interactions, as well as for diagnostic and therapeutic purposes. Single chain variable fragment antibodies (scFvs) have considerable potential for the immunological detection of small molecules, as well as proteins, in localisation of bacterial surface structures (Bradbury et al., 2011). In this study, a mix of two large human synthetic phage displayed libraries, protein-directed (Brockmann et al., 2011) and hapten-directed, was used to select scFvs against L. helveticus MIMLh5 S-layer protein. After three rounds of panning, four monoclonal scFvs capable of binding with the L. helveticus MIMLh5 S-layer protein and one capable of binding also with the S-layer protein of L. helveticus ATCC 15009T, which is different only in five amino acids, were obtained. All five identified novel anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue as alkaline phosphatase fusions (Krebber et al., 1997). ScFv-PolyH4 was used to develop an assay, based on Western blot analysis, for detection of the S-layer protein in Grana Padano cheese ripened for 2-5 months. These results showed promising applications of the scFv-PolyH4 for detecting immunologically important S-layer protein of L. helveticus in this cheese and in other food matrices and underlined the high value of the utilised phage display libraries for obtaining the extremely specific scFvs. References: Beganović, J., Frece, J., Kos, B., Leboš Pavunc, A., Habjanič, K., Šušković, J. (2011) Functionality of the S-layer protein from the probiotic strain Lactobacillus helveticus M92. Antonie Van Leeuwenhoek. 100: 43-53. Bradbury, A. R. M., Sidhu, S., Dübel, S., McCafferty, J. (2011) Beyond natural antibodies: the power of in vitro display technologies. Nature Biotechnol. 29:245-254. Brockmann, E. C., Akter, S., Savukoski, T., Huovinen, T., Lehmusvuori, A., Leivo, J., Saavalainen, O., Azhayev, A., Lövgren, T., Hellman, J., Lamminmäki, U. (2011) Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling. Protein Eng Des Sel. 24: 691-700. Guglielmetti, S., Taverniti, V., Minuzzo, M., Arioli, S., Zanoni, I., Stuknyte, M., Granucci, F., Karp, M., Mora, D. (2010) A dairy bacterium displays in vitro probiotic properties for the pharyngeal mucosa by antagonizing group A streptococci and modulating the immune response. Infect Immun. 78: 4734-4743. Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., Plückthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 201: 35-55.