Difference between revisions of "Part:BBa K525405"

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Fusion Protein of S-Layer SbpA and mCitrine

Fusion Protein of S-Layer SbpA and mCitrine

S-layers (crystalline bacterial surface layer) are crystal-like layers consisting of multiple protein monomers and can be found in various (archae-)bacteria. They constitute the outermost part of the cell wall. Especially their ability for self-assembly into distinct geometries is of scientific interest. At phase boundaries, in solutions and on a variety of surfaces they form different lattice structures. The geometry and arrangement is determined by the C-terminal self assembly-domain, which is specific for each S-layer protein. The most common lattice geometries are oblique, square and hexagonal. By modifying the characteristics of the S-layer through combination with functional groups and protein domains as well as their defined position and orientation to eachother (determined by the S-layer geometry) it is possible to realize various practical applications ([http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full Sleytr et al., 2007]).

Usage and Biology

S-layer proteins can be used as scaffold for nanobiotechnological applications and devices by e.g. fusing the S-layer's self-assembly domain to other functional protein domains. It is possible to coat surfaces and liposomes with S-layers. A big advantage of S-layers: after expressing in E. coli and purification, the nanobiotechnological system is cell-free. This enhances the biological security of a device.

This fluorescent S-layer fusion protein is used to characterize purification methods and the S-layer's ability to self-assemble on surfaces. It is also possible to use the characteristic of mCitrine as a pH indicator ([http://pubs.acs.org/doi/abs/10.1021/bm901071b Kainz et al., 2010]).

Important parameters

Sequence and Features

Expression in E. coli

Purification of SbpA fusion protein

Immobilization behaviour

After purification, solutions of monomeric SbpA S-layer proteins can be recrystallized and immobilized on silicon dioxide beads in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl₂). After the recrystallization procedure the beads are washed with and stored in ddH₂O at 4 °C in the dark. The fluorescence of the collected fractions of a recrystallization experiment with BBa_K525405 are shown in fig. X. 100 mg beads were coated with 100 µg of protein. The figure shows, that not all of the protein is immobilized on the beads (supernatant fraction) but the immobilization is pretty stable (very low fluorescence in the wash). After the immobilization, the beads show a high fluorescence indicating the binding of the SbpA | mCitrine fusion protein.

Fig. X: Measured fluorescence of collected fractions of immobilization of purified BBa_K525405 on silica dioxide beads (n = 3, 100 mg mL^-1 SiO₂, time of recrystallization: 4 h).

Optimal bead to protein ratio for immobilization

To determine the optimal ratio of silica beads to protein for immobilization, the degree of clearance ϕ_C in the supernatant is calculated and plotted against the concentration of silica beads used in the accordant immobilization experiment (compare fig. A):

The data was collected in three indipendent experiments. The fluorescence of the samples was measured in the supernatant of the immobilization experiment after centrifuging the silica beads. The fluorescence of the control was measured in a sample which was treated exactly like the others but no silica beads were added. 100 µg protein was used for one immobilization experiment. The data was fitted with a sigmoidal dose-response function of the form

with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x₀) (R² = 0.997).

The fit indicates that a good silica concentration for 100 µg of protein is 200 - 250 mg mL^-1. This set-up leads to saturated beads with low waste of protein. So a good protein / bead ratio to work with is 7 - 9 * 10^-4.

Fig. A: Degree of clearance of the fluorescence in the supernatant plotted against the concentration of silicium dioxide beads used to immobilize BBa_K525405 (n = 3). Data is fitted with dose-reponse function (R² = 0.997).

Methods

References

Kainz B, Steiner K, Möller M, Pum D, Schäffer C, Sleytr UB, Toca-Herrera JL (2010) Absorption, Steady-State Fluorescence, Fluorescence Lifetime, and 2D Self-Assembly Properties of Engineered Fluorescent S-Layer Fusion Proteins of Geobacillus stearothermophilus NRS 2004/3a, [http://pubs.acs.org/doi/abs/10.1021/bm901071b Biomacromolecules 11(1):207-214].

Sleytr UB, Huber C, Ilk N, Pum D, Schuster B, Egelseer EM (2007) S-layers as a tool kit for nanobiotechnological applications, [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full FEMS Microbiol Lett 267(2):131-144].