title: Design of a peptibody consisting of the antimicrobial peptide dhvar5 and a llama variable heavy-chain antibody fragment.
abstract: 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.
authors.affiliation: Department of Periodontology, Section Oral Microbiology, Academic Centre for Dentistry, van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Szynol
authors.forename: J J W
authors.initials: JJ
authors.lastname: de Haard
authors.forename: E C
authors.initials: EC
authors.lastname: Veerman
authors.forename: J J
authors.initials: JJ
authors.lastname: de Soet
authors.forename: A V
authors.initials: AV
authors.lastname: van Nieuw Amerongen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism*; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D002161:Camelids, New World / Q000276:immunology*; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D054808:Histatins; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012471:Salivary Proteins and Peptides / Q000235:genetics / Q000378:metabolism*
chemical_list: D000890:Anti-Infective Agents; D000906:Antibodies; C504850:Dhvar-5; D054808:Histatins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides