2766 Proteomics 2013, 13, 2766–2775 DOI 10.1002/pmic.201300009 RESEARCH ARTICLE NanoPad: An integrated platform for bacterial production of camel nanobodies aimed at detecting environmental biomarkers Sof´ ıa Fraile 1 , Jose I. Jim ´ enez 1∗ , Carlos Guti ´ errez 2 and V´ ıctor de Lorenzo 1 1 Systems Biology Program, Centro Nacional de Biotecnolog´ ıa-CSIC, Campus de Cantoblanco, Madrid, Spain 2 Department of Animal Pathology, Faculty of Veterinary Science, Las Palmas University, Canary Islands, Spain 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 (V HH ) 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, V HH 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 V HH s revealed the presence of this enzyme in the metaproteome of an oil refinery waste treatment plant. Keywords: Bioremediation / Biphenyl / BphC / Microbiology / PCBs / Western–panning Received: January 7, 2013 Revised: February 11, 2013 Accepted: March 26, 2013 1 Introduction The environmental quality of sites polluted with toxic chemi- cals relies to a large extent on the occupant microbial species as well as the abundance and diversity of their catabolic genes and enzymes [1]. The proteome of the microbial Correspondence: Professor V´ ıctor de Lorenzo, Systems Biology Program, Centro Nacional de Biotecnolog´ ıa-CSIC, Campus de Cantoblanco, Madrid 28049, Spain E-mail: vdlorenzo@cnb.csic.es Fax: +34-91-585-45-06 Abbreviations: Ap, ampicillin; BphC, biphenyl 2,3-dioxygenase; Cm, chloramphenicol; DHBD, dihydroxybiphenyl dioxygenase; Km, kanamycin; Phab, phage antibody; POD, peroxidase; RT, room temperature; scFv, single-chain variable fragment of anti- body; V HH , variable heavy-chain antibody H fragment (camelids) species that inhabit such places, and the metaproteome of the resident community as a whole, thus embodies the whole catalytic complement available for in situ remediation of the pollutants at stake [2]. The presence of given proteins in a complex mixture of environmental origin can be probed inter alia with mAbs that recognize conserved antigenic motifs of specific enzyme types [3]. The most widespread technologies to generate antibodies to this end focus their production in eukaryotic cells using hybidromas [4], genetically modified mice [5], or more recently, immortal B cells [6]. However, these methods of production are expensive and time- consuming. One alternative is the capture of the antibody ∗ Current address: Dr. Jose I. Jim´ enez, Department of Mechanical En- gineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA Colour Online: See the article online to view Fig. 2 in colour. C  2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com