Part:BBa_K525305

Fusion Protein of S-Layer SgsE and mCitrine

Fusion protein of S-layer SgsE 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

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	Inclusion body
	Compatibility	E. coli KRX and BL21(DE3)
	Induction of expression	expression of T7 polymerase + IPTG or lactose
	Specific growth rate (un-/induced)	0.139 h-1 / 0.071 h-1
	Doubling time (un-/induced)	4.98 h / 9.78 h
Purification	Molecular weight	110.2 kDa
	Theoretical pI	5.74
	Excitation / emission	515 / 529 nm
Immobilization behaviour	Saturation protein / bead ratio	5 - 7 * 10-4
	Immobilization time	4 h

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 167
  Illegal BglII site found at 1022
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 76
  Illegal AgeI site found at 3121
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1657
  

Expression in E. coli

The SgsE (K525301) gen was fused with a mCitrine (BBa_J18931) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The SgsE|mCitrine fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0,1 % L-rhamnose and 1 mM IPTG using the [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_S-layer_genes_in_E._coli autinduction protocol] from promega.

Figure 1: Growthcurve of E. coli KRX expressing the fusion protein of Sgse and mCitrine with and without induction, cultivated at 37 °C in autoinduction medium with, respectively, without inductor. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 4 h the OD₆₀₀ of the induced K525304 visibly drops when compared to the uninduced culture. Both cultures grow significantly slower than KRX wildtype.

Figure 2: RFU to OD₆₀₀ ratio of E. coli KRX expressing the fusion protein of Sgse and mCitrine with and without induction. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 4 h the RFU to OD₆₀₀ ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly four times higher. The KRX wildtype shows no variation in the RFU to OD₆₀₀ ratio.

Purification of SgsE fusion protein

As seen in the analysis of the cultivations with expression of SgsE | mCitrine fusion proteins, these proteins form inclusion bodies in E. coli. Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. So the first purification step is to solve and set-free the inclusion bodies. This step is followed by two filtrations (300 kDa UF and 100 kDa DF/UF) to further concentrate and purify the S-layer proteins. After the filtrations, the remaining protein solution is dialized against ddH₂ for 18 h at 4 °C in the dark. The dialysis leads to a precipitation of the water-insoluble proteins. After centrifugation of the dialysate the water-soluble S-layer monomers remain in the supernatant and can be used for recrystallization experiments.

The fluorescence of the collected fractions of this purification strategy is shown in the following figure A:

Fig. A: Fluorescence of collected fractions during purification of BBa_K525305 fusion protein.

A lot of protein is lost during the purification especially after centrifugation steps. The fluorescence in the urea containing fractions is lowered due to denaturation of the fluorescent protein. Some fluorescence could be regenerated by the recrystallization in HBSS. This purification strategy is very simple and can be carried out by nearly everyone in any lab being one first step to enable real do it yourself nanobiotechnology.

Immobilization behaviour

After purification, solutions of monomeric SgsE S-layer proteins can be recrystallized and immobilized on silicon dioxide beads in HBSS (Hank's buffered saline solution). 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_K525305 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 SgsE | mCitrine fusion protein.

Fig. X: Measured fluorescence of collected fractions of immobilization of purified BBa_K525305 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):

(2)

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

(3)

with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x₀) (R² = 0.874).

The fit indicates that a good silica concentration for 100 µg of protein is 150 - 200 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 5 - 7 * 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_K525305 (n = 3). Data is fitted with dose-reponse function (R² = 0.874).

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].