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Proteases play crucial roles in numerous biological processes, and understanding their functions requires biochemical characterization. A key step in the functional characterization of any protease is its overexpression and purification. However, expression of certain proteases in insect or mammalian cell-based expression systems can be difficult due to their propensity for autocleavage. Recently, advances in in vitro transcription/translation systems have facilitated the overexpression of a wide variety of proteins that had previously been challenging using other systems. Notably, the wheat germ cell-free translation system has proven highly efficient in expressing properly folded eukaryotic multidomain proteins. In this issue, Singh et al. demonstrate that the wheat germ system can also be used to overexpress intact, and functionally active, eukaryotic proteases. The authors study the high temperature requirement A3 (HtrA3) mammalian serine protease, a member of the HtrA family that is a tumor suppressor and plays a role in placental development. HtrA3 possesses a long and short isoform, with both containing a signature serine protease domain but differing in the presence of a C-terminal PDZ domain in the long isoform. Determining if these two isoforms are biochemically distinct required their overexpression, and the authors tested the feasibility of the wheat germ system for producing the two HtrA3 isoforms as intact and active proteases. When expressed at the standard temperature of 17° C used in the wheat germ translation protocol, both isoforms showed multiple bands consistent with their autocleavage. The addition of serine protease inhibitors did not help in stabilizing the synthesized protease isoforms either. However, when the authors changed from the standard temperature to 4° C, both isoforms were expressed as single bands of expected size and were shown to be enzymatically active when tested in an in vitro protease assay. This ability of the wheat germ cell-free translation system to produce intact and enzymatically active proteases should prove to be a major boon to researchers studying protease function in various fields.
Together AgainThe concept behind the bimolecular fluorescence complementation (BiFC) assay is simple: attach non-fluorescent fragments of a split fluorescent protein to either the C- or N-terminus of a pair of putative interacting proteins and if the two non-fluorescent fragments are brought together (an indication that the two proteins are interacting), the fluorescent protein will reassemble and can be detected using microscopy. In recent years, BiFC has become a valuable approach for visualizing protein interactions in living cells, especially when it comes to detecting weak protein interactions due to the stability of the fluorescent protein following reassembly. Hoping to take advantage of these strengths, Ohashi et al. at Tohoku University (Sendai, Miyagi, Japan) designed a BiFC assay to detect phosphorylation-dependent interactions between cofilin and actin. However, the authors quickly found that the fluorescent protein fragments commonly used for BiFC assays were not able to verify cofilin-actin interactions, requiring them to create a new BiFC probe. Designing fluorescent proteins to use as BiFC probes is not as easy splitting a fluorescent protein in half; it is necessary to divide the native protein at a location where it will reform its correct 3-dimensional structure when the two pairs are brought into proximity, but neither will spontaneously self assemble. In addition, the split portions of the fluorescent protein must not interfere with the native function or localization of the proteins to which they are attached. For their purposes, Ohashi and colleagues selected the fluorescent protein Venus as the basis of their new BiFC probe, owning to its strong fluorescence intensity and efficient maturation properties. They next constructed a series of expression plasmids coding for 13 N-terminal and 13 C-terminal fragments of Venus, divided within surface loops connecting 11 ß-sheets and a fluorophore-containing α-helix region in GFP-related proteins. 91 pairs of fragments were expressed in HeLa cells and it was found that 44 were able to reassemble and emit a fluorescent signal without fusion to interacting proteins. The authors then linked various Venus fragments to actin and cofilin, as well as a cofilin mutant that fails to bind actin, and screened 1,456 pairs of N- or C-terminus fused actin and cofilin constructs looking for a Venus pair possessing: (i) strong fluorescence, (ii) a lack of spontaneous self-assembly, and (iii) a distinct difference in fluorescence between cells expressing cofilin and cells expressing the cofilin mutant. In the end, one fragment pair where Venus was split at amino acid residue 210 demonstrated a clear advantage when assessing cofilinactin interactions. To further validate the usefulness of their VC210/VN210 BiFC combination, the authors detailed interactions between H-Ras and Raf1 and calmodulin and M13, demonstrating that this split Venus combination can serve as a valuable BiFC probe for visualizing interactions that cannot be detected using the previously reported Venus fragments combinations.
