Cloning and Expression of the eIF4G subunit of Arabadopsis eIF4F

Lauren Harkinson
University of Texas at Austin

eIF4G is a eukaryotic polypeptide chain initiation factor that acts as a scaffolding protein to bring together the initiation complex that allows the 40S ribosomal subunit to bind to the mRNA. eIF4F contains subunits eIF4G and eIF4E, and plants have two forms of eIF4F; eIF4F and eIF(iso)4F. Not much is known about the difference between the two forms of eIF4F, but only plants seem to have eIF(iso)4F. Since agriculturally devastating plant viruses have been known to hijack these initiation factors to force the cell to produce viral proteins, some plants that have viral resistance have been shown to have knockouts in various subunits of eIF4F or eIF(iso)4F. Arabadopsis thaliana is a flowering plant in the mustard family whose genome has been sequenced, and is an excellent model system for plants because it can be used for genetic studies. The entire eIF4G gene and a truncated version that was created to optimize expression have been cloned into vectors for protein expression in bacteria.
The truncated eIF4G component of eIF4F is being expressed in BL21 E. coli cells. The eIF4E component of eIF4F was previously cloned and is being expressed in BLR E. coli cells. Both types of E. coli cells are grown separately in culture and then combined, so that when the cells are sonicated to break them open, both truncated eIF4G and eIF4E will be present and can bind to each other in solution. By allowing this binding event to take place, the eIF4F complex is stabilized and less subject to degradation. Since truncated eIF4G is a very large protein, degradation is a major concern. A new broth, containing twice as many nutrients as usual (2x LB), was accidentally used and approximately doubled the amount of protein expressed per gram of cells. The eIF4F is purified after sonication by cation exchange, since eIF4G is positively charged in solution. Then affinity chromatography easily purifies the mixture by utilizing eIF4E’s job in translation, which is to bind the 7-methyl-GTP cap on the 5’ end of the mRNA. The expressed protein is being used to raise antibodies against recombinant Arabadopsis eIF4F. The Browning lab is in the process of creating Arabadopsis knockout plants in these genes of interest, and the eIF4F antibodies will be crucial in determining protein levels in the plants through Western blot analysis. These antibodies as well as the purified protein can be used for further studies to differentiate between the roles of eIF4F and eIF(iso)4F in initiation of translation in plants.