Developing antibodies to Arabidopsis thaliana translation initiation factor eIF4G

Abstract

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 for more stable folding of the protein. However, no large differences in expression were seen. Different growth media were compared in protein expression levels, using media that slowed expression over a long amount of time and allowed for better folding and stabilizing of the protein, or media that provided excess amounts of nutrients to increase the output of all proteins, or media that simply followed basic protocols. The slow-expression medum did not result in more final protein. Conversely, the excess-nutrient medium resulted in an increase in eIF4G protein, but also an even larger amount of contaminant proteins that were not the desired eIF4G. Compared to the basic media, there was not a significant increase in yield of pure eIF4G. Lastly, because the full-length eIF4G protein is approximately 150 kD, the excessive length of an already unstable protein could explain the surprisingly low protein expression level. A cDNA for an N-terminal truncation of eIF tr4G (~ 24 kDa) was prepared. This construct has a His-tag on the Cterminus to simplify purification. Antibodies raised to the N-terminal portion of eIF4G will presumably react with the full-length protein. From this shortening of eIF4G, a noticeable increase in protein expression was observed. From purified eIFtr4G protein, antibodies were produced in rabbits. This eIF4G-specific antibody can be used in the future to determine the presence of eIF4G protein or eIF4F complex, and can serve to clarify their roles in certain phenotypes and biochemical pathways.