Prokaryotic Expression, Purification, and Polyclonal Antibody Identification of Chlamydomonas reinhardtii IFT20

Abstract:

Intraflagellar transport protein 20 (IFT20) of Chlamydomonas reinhardtii is a component of the intraflagellar transport complex B(IFT-B). IFT20 is known to be involved in regulating intracellular transport processes of macromolecules, but its detailed function as a flagellar protein in flagella remains to be investigated. This study aimed to develop highly specific polyclonal antibodies against Chlamydomonas reinhardtii IFT20 from rabbit serum to lay the foundation for further investigation into its flagellar function. To achieve this goal, the N-terminal 6×His-tagged IFT20 fusion protein(6×His::IFT20) was first expressed in Escherichia coli, and 6×His::IFT20 was purified by Ni-column. The purified 6×His::IFT20 was then applied to immunize the New Zealand white rabbits. The antiserum was collected after three rounds of immunization, and the titer was determined to be 1∶102 400 by an indirect ELISA method. Subsequently, the obtained anti-serum was purified using protein A purification beads to enrich the IgG subtype antibodies. Next, N-terminal MBP-tagged IFT20(MBP::IFT20) expressed and purified from Escherichia coli was used for antigen-antibody affinity purification of IgG antiserum. The western blotting assay of whole cell protein extracts from CC-125 identified the high specificity of the obtained IFT20 polyclonal antibodies, making them suitable for future investigations on the flagellar function of IFT20 in Chlamydomonas reinhardtii.