Construction Of Anticalin Library And Screening Antiestradiol, Antiparaquats By Ribosome Display

In this dissertation, ribosome display method was developed for determination oftwo typical pollutants of environment, estradiol （E₂） and paraquats （PQ）. Principles andtechnologies of selection for anticalin analysis and monitoring were also presented.Estradiol （E₂） and its derivatives, which can promote animal growth and improvelean meat proportion in livestock and poultry, also have a wide range of pharmacologicalapplications. The development of soluble receptor proteins that recognize E₂is becomingincreasingly important in biotechnology and life sciences research.Paraquat （PQ） is a bisquaternary ammonium compound used as herbicideword-widely. There is a potential for human exposure to PQ in general populationthrough residues on food materials and water.However actual methods, such as GC,HPLC and UV, are time consuming and need expensive instruments. Thus, developmentof a rapid and convenient detection method for local PQ analysis is extremely desirable.For the past century, this field of application has been dominated by antibodieswhich were traditionally generated through immunization of animals. Antibodies are themost widely used class of reagents for research, pharmaceutical, diagnostic, andtherapeutic applications. However, the preparation of anti-hapten antibodies requiresvarious skillfully executed experiments and takes a long period of time. Recently, a farmore effective alternative display system based on ribosome has been reported. Thistechnology relies on non-covalent ternary polypeptide-ribosome-mRNA complexes,which ensure the coupling of genotypes and phenotypes. These complexes, which areformed during in vitro translation, can be directly used to select the properties of thedisplayed protein. Based on the foregoing, the applicability and efficiency ofcommercially available reconstituted translation systems in ribosome display andselection of functional proteins were investigated in the present study.Lipocalins normally function as storage and as the means of transport ofphysiologically important compounds. Lipocalins form a large family of extracellularproteins.Despite large diversity at the sequence level, they share a conserved barrel ofeight antiparallel β-strands with an attached α-helix as their scaffold. At one end of thebarrel, four loops connect each pair of strands and form the entrance to the bindingpocket that determines the specificity of the ligand.20Based on this notion, the use oflipocalins, in particular the bilin-binding protein （BBP）, for the construction of a novelclass of receptor proteins termed ‘anticalins’has been established. In the present study, we have made another attempt to reshape the BBP with itsrather wide and shallow ligand pocket to recognize estradiol （E₂） and paraquat （PQ） asligand. The conjugates between one of the estradiol and paraquat derivatives and certaincarrier proteins have been used to select a cognate anticalin from a combinatorial libraryof BBP mutants by the combined use of the ribosome display technique. In the currentresearch, anticalin specifically binding with E₂and PQ was chosen after several rounds ofselection.Because of the versatility of native antibody library, specific antibody can beselected for any kinds of antigen theoretically. This methods established the foundationthat we can select specific antibodies of any antigens especially micromolecule haptens.