Difference between revisions of "Part:BBa K525234"

Revision as of 23:45, 21 September 2011

Fusion Protein of mRFP, S-layer cspB from Corynebacterium halotolerans with TAT-sequence, PT7, RBS

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.

Important parameters

Sequence and Features

Expression in E. coli

The CspB gen was fused with a monomeric RFP (BBa_E1010) using [http://2011.igem.org/Team:Bielefeld-Germany/Protocols#Gibson_assembly Gibson assembly] for characterization.

The CspB|mRFP fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0,1 % L-rhamnose 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 CspB and mRFP 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 6 h the OD₆₀₀ of the induced K525224 visibly drops when compared to the uninduced culture. While the induced culture grow significantly slower than KRX wildtype the uninduced seems to be unaffected.

Figure 2: RFU to OD₆₀₀ ratio of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 6 h the RFU to OD₆₀₀ ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly seven times higher at its highest point but starts to drop during the cultivation due to degradation of the fusion protein. Most likely due to basal transcription the RFU to OD₆₀₀ ratio of the uninduced culture starts to rise after 12 hours. The KRX wildtype shows no variation in the RFU to OD₆₀₀ ratio.

Intracellular localisation

Purification

After the localisation of the S-layer protein in E. coli, different methods for purification were tested. The results of these methods are shown in fig. X. Fig. X shows, that the CspB protein does not form inclusion bodies in E. coli and most of the protein is transported out of the cell into the periplasm and a lot of protein is even secreted into the medium (all fractions were concentrated by filtration and precipitation, respectively). The secretion into the culture medium is very interesting because the purification is much faster (no cell disruption necessary).

Fig. X: Fluorescence of collected fractions of different methods to release and concentrate BBa_K525234 protein from a cultivation in E. coli.

The highest fluorescence could be obtained by a precipitation with ammonium sulfate of the culture supernatant followed by an ultrafiltration with a 300 kDa membrane and a diafiltration with a 50 kDa membrane. The diafiltration was against a binding buffer for an anion exchange chromatography (25 mM sodium acetate, 25 mM sodium chloride) with pH 6, due to the theoretical pI of BBa_K525234. The fluorescence of the collected fractions of the following anion exchange chromatography are shown in fig. B.

Fig. B: Fluorescence of collected fractions of an anion exchange chromatography of BBa_K525234 after concentration from the culture supernatant.

The binding conditions are well chosen because nearly all of the protein binds to the column. The protein is eluted from the column with rising sodium chloride concentrations. The highest fluorescence is in the elution fraction with 400 mM sodium chloride. 600 mM sodium chloride elutes all of the S-layer fusion proteins.