Abstract
With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated “silencing” represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein–protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.
Highlights
The approach of choice for elucidating biological mechanisms is to eliminate the functions of biomolecules and to observe the responses of the organism
Liquid yeast cultures were harvested and analyzed by western blot to determine the correct expression of the ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS (AHP) and recombinant antibody fusion proteins with Gal4 DNA activation domain (AD) and Gal4 DNA binding domain (BD), respectively (Figure S1 and Figure S2)
Recombinant antibodies were tested in this assay against all six highly homologous AHP proteins (Figure 2A)
Summary
The approach of choice for elucidating biological mechanisms is to eliminate the functions of biomolecules and to observe the responses of the organism. A targeted application of specific inhibitors in order to block a selected biomolecule function allows better control over the process. Biomolecular interactions constitute the core of biological functions, and these can be defined by their specificity and affinity. Many proteins have evolved into extraordinarily precise biomolecules and can be exploited as specific inhibitors of biomolecular interactions. Antibodies stand out for their unique ability to recognize with relatively high specificity and affinity a virtually unlimited number of target biomolecules, known as antigens. This capability makes antibodies truly indispensable tools in biological research today. Such tools can be used for antibody-mediated protein ‘‘silencing’’
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
