Developing antibodies to Arabidopsis thaliana translation initiation factor eIF4G

In eukaryotes, a key way to regulate biochemical pathways and protein synthesis is through translation, the reading of an mRNA sequence to make proteins and enzymes. In translation, one of the main steps that can be regulated is the formation of the initiation complex, which is necessary to position the machinery at the right spot on the mRNA before protein can be generated. A major player in translation initiation is the eIF4F complex (eukaryotic initiation factor 4F), made up of three different proteins: eIF4A, eIF4E and eIF4G. The eIF4F complex specifically functions to recruit the small ribosomal unit, which serves as an interface between mRNA and the new protein being made, to the correct place on the existing mRNA strand. In this way, eIF4F may control the rate of protein synthesis and, ultimately, the abundance, or conversely the absence, of specific proteins in organisms. Therefore, the eIF4F complex may be useful in controlling expression systems, which can range in application from hormone production in animals to synthesizing useful medicines in plants. One of the major goals of the Browning lab is to understand better the effects of eukaryotic initiation factor mutants on phenotype in the Arabidopsis thaliana plant. One part of the lab research thus focuses on the generation of " knock out " A. thaliana mutants, eliminating one or a combination of initiation factors. Antibodies specific to the different initiation factors are useful for assaying protein expression and for immunolocalization, which allows you to visualize the presence or location of the protein which bind to that antibody. While antibodies have been generated for several initiation factors already, some still remain elusive. In a previous project, I produced antibodies to the Arabidopsis eIF4F complex and the lab already possesses Arabidopsis (At) eIF4E antibody. However, antibodies specific to AteIF4G have not yet been made, due to difficulties 4 producing ample AteIF4G protein for antibody generation. It is important to differentiate between Arabidopsis and wheat initiation factors, as the possessed wheat eIF4G antibody does not cross-react with AteIF4G. A large part of the problem may be attributed to A. thaliana eIF4G protein instability compared to wheat eIF4G; the protein is prone to rapid degradation and low expression levels in E. coli. In order to optimize the expression of AteIF4G, different vectors, expression systems, growth media, bacterial growth conditions and protein size were tried. In testing different vectors and expression systems, I looked …