Screening of a full synthetic VHH antibody library against recombinant enterokinase through phage display technology.
نویسندگان: Pedram Rezaei Amirkiasar, Dr. Mahdi Aminian ©
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Background and Aim
Mammalian Enterokinase (EC 22.214.171.124) is a duodenal digestive enzyme which has a two-subunit glycoprotein structure and contains a single disulfide bond. Moreover, enterokinase exhibits a remarkable specificity towards the (Asp)₄ -Lys sequence inside the protein structures. Hence, it is widely considered as a biotechnological tool in the digestion of fusion proteins and elimination of purification and reporter tags such as poly His, GST, and GFP. In the present study, we aimed to develop novel recombinant monoclonal antibodies against enterokinase enzyme by the screening of a Camelidae VHH library through phage display technology. The isolated antibodies are to be chemically coupled on a chromatographic media as the affinity ligands for purification purposes.
In this study, we performed the phage display technique through several consecutive biopanning sessions to screen the recombinant nanobodies specific to recombinant enterokinase by the ELISA method. Furthermore, the specificity and affinity of the antibodies against recombinant enterokinase were evaluated using polyclonal phage ELISA. Eventually, the monoclonal phage ELISA has been performed as well.
The specificity and affinity of the selected antibodies were evaluated following the biopanning process. According to data obtained from polyclonal phage ELISA, a distinctively significant difference was observed between the absorbance density of the coated wells and the control ones by about 2 units. In the next step, the eluted phages obtained from round 5 of the biopanning process were used to perform monoclonal phage ELISA and thereby a single VHH-phage clone was isolated which was tightly bound to the surface epitopes of the enterokinase enzyme.
According to the results obtained in this study, the Camelidae VHH library demonstrates efficient specificity and also high binding capacity towards the enterokinase enzyme therefore could act as an effective affinity ligand to be chemically coupled on a chromatographic media for purification purposes.
Enterokinase; Phage display; VHH library; Biopanning; Specificity; Phage ELISA
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