SslA truncation derivatives

Mutagenesis and molecular characterization of SslA truncation derivatives... 

S.3.2 Heterologous expression in E. coli, isoiation and purification of SslA truncation derivatives... 

After the successful expression and purification of the SslA truncation derivatives, their self-assembling properties and lattice symmetry formation were investigated. Are these derivatives able to self-assemble in vitro or is this ability lost with certain amino acid deletions If self-assembly is preserved, what do the selfassembling structures look like Can the p4 symmetry, as in case of the native SslA, be observed Which factors will influence the self-assembly process To answer all these questions, in vitro recrystallization experiments were conducted on a silicon wafer. 

For investigating the self-assembly properties of the SslA truncation derivatives on a solid substrate, the proteins were recrystallized on a functionalized Si surface at a protein concentration of 0.5 mg/ml. In particular, the purified S-layer solutions were at first dissolved in 5 M GuHCl and one-fifth of the solution dialyzed against Tris/HCl buffer pH 3 for 2 h at 4 °C in the presence of an APTES functionalizes Si substrate. After dialysis, the Si substrate was washed with distilled water, dried on air and analyzed by AFM. For the N-terminal tmncation derivative the resulting self-assembly structures are shown in Figure 3.13. 

Figure 3.13 shows that the N-terminal SslA truncation derivative was able to self-assemble under the above-mentioned in vitro recrystallization conditions. The resulting self-assembly structures take the form of crystalline protein multilayers extending to sizes between 1 and 3 pm (Figure 3.13a and c). A 3D reproduction of the multilayer stmcture is shown in Figure 3.13d. The thickness of one layer is approximately 3 nm, as indicated by the cross-section profile displayed in Figure 3.9b. A similar height was measured for this derivative when recrystaUized in solution.

The N-terminal SslA truncation derivative (SSIA341.1097N)... 

Self-assembly of the recombinant SslA truncation derivatives on a Si wafer. 

In conclusion, the SslA truncation derivatives created have kept their ability to st i-assemble in vitro in solution and on a Si wafer. The proteins formed crystalline nanostructures that, depending on the application, can be considered promising building blocks for nano(bio)technology. Furthermore, the recrystallization experiments have demonstrated that the central SslA protein part is sufficient for self-assembly and for the square lattice symmetry formation. The result opens up new possibilities for further genetic modifications of this recombinant S-layer toward interesting applications in nano(bio)technology. 

The C-terminal truncation derivative of the recombinant S-layer of S. ureae ATCC 13881 was obtained by cleavage of the last 172 amino acids of the recombinant SslA protein sequence and named SSIA32.925C. The gene encoding this SslA derivative consists of 2724 bps, which translates into an S-layer protein of 907 amino acids with a molecular weight of 97 kDa and pi of 5.46. The final protein stmcture additionally contains a Hisg tag. 

Based on the comparison of these sequence data, truncations lacking the N-terminal 341 amino acids and the C-terminal 172 amino acids were created plus a derivative lacking both of these SslA protein parts. The design of these derivatives is depicted in Figure 3.11. 

Truncation analysis of SbsC revealed that the C-terminal 179 amino acids are not required for the formation of the oblique lattice structure. In the case of this smallest SslA derivative, the C-terminal 172 amino acids were deleted and the... 

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