title: NanoPad: an integrated platform for bacterial production of camel nanobodies aimed at detecting environmental biomarkers.
abstract: The presence of given antigens in environmental samples (e.g. biodegradative enzymes) reports the quality and catalytic vigor of particular soils or aquatic ecosystems. In this context, we have developed the NanoPad system consisting of a complete platform for isolation, amplification, and extracellular production of specific antibodies against antigens that diagnose the occurrence of protein markers in crude environmental samples. The workflow starts with the inoculation of camels (Camelus dromedarius) with various proteins (e.g. catabolic enzymes) for generating a phage display library of variable heavy-chain antibody H fragment (VHH ) domains that bind the different antigens. Instead of being subjected to a conventional panning, such a library is then probed with a Western-panning technique that allows direct isolation of specific binders of proteins blotted on membranes from polyacrylamide gels. Finally, VHH s are fused to the C-domain of hemolysin for secretion to the culture media as virtually pure dimeric proteins that can be used as a primary antibody without further processing. The value of NanoPad is shown with the selection of nanobodies for detection of biphenyl 2,3-dioxygenase, a key enzyme for biodegradation of polychlorinated biphenyls. The thereby generated anti-biphenyl 2,3-dioxygenase VHH s revealed the presence of this enzyme in the metaproteome of an oil refinery waste treatment plant.
authors: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid, Spain.
authors: Sofía
authors: S
authors: Fraile
authors: Jose I
authors: JI
authors: Jiménez
authors: Carlos
authors: C
authors: Gutiérrez
authors: Víctor
authors: V
authors: de Lorenzo
mesh_terms: D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis*; D015153:Blotting, Western; D002162:Camelus / Q000378:metabolism*; D049308:Dioxygenases / Q000378:metabolism; D004783:Environmental Microbiology; D004797:Enzyme-Linked Immunosorbent Assay; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D015202:Protein Engineering; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000096:biosynthesis*
chemical_list: D015415:Biomarkers; D007143:Immunoglobulin Heavy Chains; D020543:Proteome; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase
keywords: Bioremediation; Biphenyl; BphC; Microbiology; PCBs; Western-panning