Three Strategies For Nanobody Expression In E.coli And It Functional Characterization

Camelid single-domain antibodies(sdAbs,VHHs,or NanobodiesR)are kinds of antibody fragments that are composed of the heavy-chain variable domain only.They have many characteristics,such as they are small in size and exhibit thermal stability and high solubility.Compared to conventional antibodies,VHHs can be manufactured in microorganisms to significantly save on cost,labor,and time since VHHs lack the Fc domain with its N-linked oligosaccharide.Until now,VHHs have been expressed in several kinds of production systems,ranging from prokaryotic cells,yeasts,fungi,insect cells,and mammalian cell lines,to plants.In thesis,we chose E.coli as the expression system of VHH2 and used 3 methods to obtain the expected proteins.Firstly,we obtained only 3 mg/L yield of VHH2 through periplasmic expression.After that,we constructed a fusion protein which fused VHH2 with the disulfide-isomerase DsbC,8 mg/L DsbC-VHH2 could be obtained.Then,we cleaved the DsbC-VHH2 by enterokinase and collected VHH2 by IMAC.Lastly,we directly expression VHH2 in the cytoplasmic space of an E.coli double mutant strain.Yield of 17 mg/L VHH could be obtained after simple optimization.Besides,we got good results from this method for expression another two VHH sequences.Then,we tested the function of VHH2 by western blot,ELISA,qPCR,SPR,cell ELISA and immunofluorescence.In thesis,three expression strategies have been compared.For the first time,we used mutant stain to express VHH and proved that VHH expressed in mutant stain is better than periplasm.This expression method could be used for a variety of VHH sequences.Besides,the fusion tag DsbC increase the yield significantly and this result may help the researches of the relationship between DsbC and VHH.