Team:Slovenia/Regulated assembly Results.html
The genes encoding protein domains were fused according to our BioBrick standard, and produced by fermentation in E.coli BL21(DE3) pLysS.
Figure 1: Flow chart of the protocol for GyrB-foldon and GyrB-CutA1 isolation.
GyrB-foldon protein was expressed in the soluble fraction. Cell lysate was further purified on Ni-NTA column resulting in purified GyrB-foldon. Expression and purification efficiency were confirmed by SDS-PAGE (Figure 7).
Figure 2: SDS PAGE demonstrating the production and purification of GyrB-foldon. Calculated size of this polypeptide is 29 kDa. Lane 1 – MW standard, lane 2 – GyrB-foldon in cell lysate supernatant, lane 3 – GyrB-foldon in insoluble fraction, lane 4 – GyrB-foldon purified by chelating chromatography.
Expression was confirmed also with western blot analysis using primary mouse anti-His-tag antibodies and secondary goat anti-mouse antibodies conjugated with horse-reddish peroxidase (HRP). In addition a strong band is seen at the position of a trimer, suggesting very stable trimeric interaction (Figure 8, line 2).
Figure 3: GyrB-foldon expression confirmed by western analysis using primary anti-His-tag antibodies for detection. GyrB-foldon is predominantly expressed in the soluble fraction. Lane1 – standard, lane 2 – GyrB-foldon in cell lysate supernatant.
We also produced a GyrB-CutA1 fusion protein consisting of N-terminus GyrB domain and CutA1. CutA1 is another protein which forms trimers. We used CutA1 from E. coli, which takes part in copper metabolism in bacterial cells (Caldrone et al., 2003). Aggregation of CutA1 domain is induced upon addition of divalent metal ions. Due to GyrB domain this fusion protein can assemble upon the addition of coumermycin. Coumermycin crosslinked samples were examined with transmission electron microscopy (Figure 9). On Figure 9 the porous material is visible. GyrB-CutA1 model predict that hexahedrons have a diameter of 23,48 nm (Figure 5A). This construct is described into more detail in the Registry of parts (BBa_K245050). After fitting our model on TEM picture we can find pores of predicted size (Figure 9).
Figure 4: Porous structure of the material consisting of GyrB-CutA1 fusion protein crosslinked by coumermycin. examined by TEM. Molecular models of gyrB-CutA1 hexamer units are shown in scale for comparison (cyan).
Planar hexagonal lattice is not the only possible structure that could be formed. Assembly of trimeric-dimeric polypeptide could also produce closed polyhedra that join three edges at their vertexes, such as tetrahedron, cube, dodecahedron, »bucky ball« (Buckminster fullerene) and others. Similar as for the assemblies based on coiled-coil segments, the size and flexibility of linker hinges as well as polypeptide concentration will determine the type of assembly. In addition the position of connection between trimeric and dimeric doimain affects the geometry of the assembly. Note that in case of foldon the N- and C-terminal ends are in close proximity (0.7 to 1.3 nm), while in CutA1 the ends are separated by 3.4 nm and 4.3 nm at the N- and C-terminus respectively, which may favor formation of polyhedra with acute angles versus planar packing for foldon, respectively. Here we expect to find the best use of our BioBrick standard and vector which allows us to lengthen the linker between polypeptide domains to the desired length. This kind of closed assemblies could be used as nanocages to enclose compounds, which are larger than the size of pores. Their most promising potential may be in drug delivery as the enclosed compounds could be released into the organism under conditions which disassemble dimeric (or trimeric) segment.
In conclusion we successfully prepared and tested DNA constructs coding for fusion proteins composed of oligomerization domains that can form the porous material. The main advantage of this material is that its assembly and disassembly can be controlled. Gyrase B subunit and combination of coumermycin and novobiocin addition makes this system convenient for the regulated assembly/disassembly of the material. In addition to gyrase B/coumermycin combination there are several other similar known protein domains, such as for example immunophilins/tacrolimus (FK506).