Watching Too Much TEV Is Bad for Your Flies

A major advantage of Drosophila for the study of gene function has been its amenability to analysis by traditional genetics, and this has been recently augmented by various powerful molecular biological techniques such as the Gal4/UAS binary expression system, FLP/FRT conditional mitotic recombination, and RNA interference. These widely used methodologies are unsuitable, however, for studies requiring the direct intracellular manipulation of a target protein since they affect gene expression at the transcriptional or translational level. Only a few approaches have been developed in Drosophila to directly affect proteins in vivo, such as conditionally splicing inteins or FlAsH-FALI photoinactivation. In this issue, B. Harder and colleauges in the laboratory of R. Schuh at the Max Planck Institute for Biophysical Chemistry (Göttingen, Germany) have devised a method for the targeted intracellular cleavage in Drosophila of proteins engineered with the peptide recognition sequence of the tobacco etch virus (TEV) protease. This protease recognizes the seven-peptide consensus sequence E-X-X-Y-X-Q-G/S and the canonical sequence E-X-X-Y-X-Q-G/S, which is the most efficiently cleaved and is routinely used for the in vitro removal of epitope tags from recombinant proteins. Harder et al. first demonstrated that a Tao protein kinase engineered with a TEV recognition sequence could be cleaved in Drosophila tissue culture cells. They then determined that ubiquitous or tissue-specific expression of TEV protease in whole flies affected neither their viability nor fertility. When TEV protease and the same engineered Tao protein kinase were co-expressed in embryonic tracheal cells, the Tao protein was also cleaved specifically in those cells. Finally, and most significantly, they were able to phenocopy a mutation in the gene for the Drosophila claudin megatrachea with their system. A transgene of the Megatrachea protein with a TEV site separating the essential C-terminal domain from the rest of the protein and expressed under the control of the endogenous mega regulatory sequences was first shown to fully rescue the mega null mutant phenotype of malformed embryonic trachea. When TEV protease was expressed in the tracheal cells of these rescued flies, a phenocopy of the mega mutant trachea resulted, clearly demonstrating that TEV protease cleavage could be used to directly interfere with Drosophila protein function in vivo.

(See “TEV protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms” on page 765.)

Suppression of Inclusion by Fusion

Bacterial expression systems are often used for the synthesis and purification of recombinant proteins. Although this technique has been successfully used for numerous proteins, it may not be efficient for other proteins due to complications such as poor expression, low yield, instability, and difficulty in purification. Expression of certain proteins in Escherichia coli can often result in the accumulation of insoluble aggregates, forming inclusion bodies. Therefore, it is important to design expression strategies, such as using fusion proteins, to improve the production of soluble proteins. Prompted by a recent description that some thermostable proteins improve the stability and purification of target proteins fused to them, Thapa and colleagues in the lab of II-Seon Park at Chosun University (Gwanju, Republic of Korea) have done a systematic screen for E. coli thermostable proteins capable of enhancing the solubility of target proteins fused to them. To isolate thermostable proteins from the E. coli proteome, the soluble fraction of a whole-cell lysate was boiled, and the soluble fraction from this was subjected to 2-D PAGE. Protein spots from this gel were identified by mass spectrometry, and the authors then selected 10 proteins for cloning, expression, and testing of overexpression and thermostability. Among these proteins, trigger factor (TF)—a 48 KD E. coli molecular chaperone involved in protein folding—was selected as the most suitable fusion partner since it was expressed well and was mostly soluble. As test target proteins, amyloid β_(1-42) peptide (Aβ_(1-42)) or a reverse form of caspase-2 (reverse-caspase-2) were chosen because they were known to form inclusion bodies in E. coli, and trials to recover soluble and active protein had not been successful. For this fusion system, a plasmid vector was constructed containing TF with a ubiquitin sequence to allow specific cleavage of the target protein from TF by the deubiquitylating enzyme Usp2-cc, and Aβ_(1-42) or reverse-caspase-2. The authors demonstrated that these target proteins can be produced and recovered in a soluble state with satisfactory yields using easy purification steps and specific cleavage of the fusion protein. These improvements, therefore, make this technique a promising tool for the efficient production of proteins and peptides that are normally insoluble and form inclusion bodies in E. coli.