A study of fine structural details of a lipopolysaccharide-binding single domain antibody selected from phage display library
Abstract
Gram negative sepsis caused by endotoxin incurs huge economic burden in human and animal health sector, globally. Various rational therapies have been attempted in past decades with futile outcomes. Endotoxin neutralisation have been attempted with antibodies targeting the core and O polysaccharide regions as epitope with some success in experimental studies. Recombinant single domain antibodies produced by phage display technology provide newer therapeutic candidates to target challenging epitopes with improved affinities. The objective of present study were to determine the affinity of a dAb clone 26 with LPS and also to biophysically characterize the dAb clone 26. In the present study, LPS was extracted and purified from an isolate of E.coli (ATCC25922). The dAb was expressed with C-terminal 6xHis tag in VHH clone 26- pET303 transformed BL21(DE3) under IPTG induction and monitored with SDS-PAGE profile. The dAb clone 26 was purified with Ni-NTA affinity chromatography. The silver stained dAb clone26 band was subjected to MALDI-TOF/MS analysis after the in-gel trypsin digestion and the partial sequence was obtained. The affinity of dAb clone 26 with LPS was analysed in SPR experiment which revealed a binding constant KA of 8.1x106M-1. Further, the dAb clone 26 was subjected to co-crystallisation with grid screening approach in a hanging drop vapour diffusion set up in 96 different conditions with pH and precipitant(Ammonium sulphate and PEG-3350,6000& 8000) as variables. The cocrystallisation resulted in crystal hits in three different condition- one each in ammonium sulphate, PEG3350 and PEG8000. In conclusion, the sequence of dAb clone 26 has been verified whereas the affinity and crystallisation experiments have scope of further optimisation.