Part:BBa_K525222

S-layer cspB from Corynebacterium halotolerans

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.

Important parameters

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	Cell membrane
	Compatibility	E. coli KRX
	Induction of expression	L-rhamnose for induction of T7 polymerase
	Specific growth rate (un-/induced)	0.245 h^-1 / 0.109 h^-1
	Doubling time (un-/induced)	2.83 h / 6.33 h

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 1103
Illegal XhoI site found at 559
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 226
Illegal NgoMIV site found at 1315
Illegal AgeI site found at 217
Illegal AgeI site found at 458
Illegal AgeI site found at 505
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 907
Illegal BsaI.rc site found at 214
Illegal BsaI.rc site found at 592
Illegal BsaI.rc site found at 994

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 mRFP|CspB 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 K525222 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 five times higher. 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.

Identification and localisation

After a cultivation time of 18 h the mRFP|CspB fusion protein has to be localized in E. coli KRX. Therefor a part of the produced biomass was mechanically disrupted and the resulting lysate was wahed with ddH₂O. Then the lysate was treted with ionic, nonionic and zwitterionic detergents to release the CspB|mRFP out of the membranes, if it intigrates. From the other part of the cells the periplasm was detached by using a osmotic shock.

The fluorescence in all cultivation fractions plus the fluorescence in the lysis und wash fraction shows that the fusion protein is water soluble and sediment not during centrifugation. Together with the absence of flourescence in the detergent fractions verifies that the fusion protein is not integrated into the cell membrane (fig. 3) and is not present as inclusion body.

In comparison with the mRFP fusion protein of ???, wich has a lipid anchor, a minor relative fluorescence per OD₆₀₀ in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD₆₀₀ after 14 h of cultivation (fig. 2) indicates this rusult a postive effect of the lipid anchor on the protein stability.

Figure 3: Fluorescence pro OD₆₀₀ progression of the mRFP (BBa_E1010)/CspB fusion protein initiating with the cultivation fractions up to the detergent fractions of the seperate denaturations. Cultivations were carried out in autoinduction medium at 37 ˚C. The cells were mechanically disrupted and the resulting biomass was wahed with ddH₂O and resuspendet in the respective detergent. The used detergent acronyms stand for: SDS = 10 % sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % n-lauroyl sarcosine; 2 % CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

MALDI TOF analysis was used to identify the location of the fusion protein in different fractions. Fractions of medium supernatant after cultivation, periplasmatic isolation, cell lysis and following denaturation in 6 M urea were loaded onto a SDS_PAGE. After denaturation with 6 M urea the remaining pellet (after centrifugation 15,000 g for 30 min) was washed with 2 % (v/v) Triton X-100, 2 % SDS (w/v). This fraction was also loaded onto the SDS-PAGE and fragments of the gel were measured with MALDI TOF.

Figure 4: SDS-PAGE of CspB/mRFP (BBa_E1010) fusion protein. Lanes are fractions of medium (M), periplasmatic isolation (PP), cell lysis (L), denaturation (D) and wash with Triton X-100 (T). Used Marker is PageRuler ^TM Prestained Protein Ladder SM0671. Marked regions were cut out and prepared for MALDI TOF analysis.

The following table shows the sequence coverage (in %) of our measurable gel samples with the amino acid sequence of fusion protein CspB/mRFP (BBa_E1010).

number of gel sample	sequence coverage (%)
1	0.0
2	0.0
3	0.0
4	0.0
5	0.0
6	0.0
7	0.0
8	0.0
9	0.0
10	0.0
11	0.0
12	14.6
13	9.3
14	6.3
15	0.0
16	0.0
17	0.0
18	0.0
19	0.0
20	1.0
21	0.0
22	0.0
23	0.0
24	0.0
25	0.0

Fig. 5 shows these data. The gel samples were arranged after estimated molecular mass cut out from the gel.

Figure 5: MALDI TOF measurement of CspB/mRFP (BBa_E1010) fusion protein. Samples are arranged after estimated molecular mass of the gel slice. Measurement was performed with a ultrafleXtreme^TM by Bruker Daltonics using the software FlexAnalysis, Biotools and SequenceEditor.

As expected, no sequence coverage was found in the periplasmatic fraction, due to absent tat sequence located at the amino-terminus. Little fluorescence was also found in the fraction of supernatant of the media, indicating that the protein can not be transported to the periplasm and thus secretion into the medium does not take place. The denaturation fraction and the Triton X-100 fraction show no or very few sequence coverage, however the lysis fraction shows significant higher sequence coverage. Both results indicate, that the fusion protein solely present in the cytoplasm and thus only identified in the lysis fraction. Fig. 5 and FIg. 6 show that the protein can be found mainly in the lysis fraction, but in smaller amounts ind the periplasmatic and the media fraction, which can be explained due to the abscence of the lipid anchor. The anchor normally binds to the cell membrane, so no protein is found in other than the lysis fraction.

To obtain more specific informations about the location of the S-layer fusion protein, after comparison with same treated fraction of E. coli KRX all gel bands in a defined size area were cut out of the gel and analysed with MALDI-TOF. Results are shown in fig. 6.

Figure 6: MALDI-TOF measurement of CspB/mRFP (BBa_E1010) fusion protein in different fractions. Abbreviations are Ma: Marker (PageRuler ^TM Prestained Protein Ladder SM0671), M (medium), PP (periplasm), L (cell lysis with ribolyser), W (wash with ddH₂O). In the left half of the gel fractions of E. coli KRX with induced production of fusion protein, the right half shows fractions of E. coli KRX without carrying the plasmid coding the fusion protein. Colours show the sequence coverage of the gel lane, cutted out of the gel. Colours mark the sequence coverage measured with MALDI TOF. fractions M of K525232 and E. coli KRX were switched and are therefore special named.

MALDI TOF measurement shows sequence coverage in the supernatant fraction of the medium, the periplasmatic fraction and the lysis fraction, indicating that the fusion protein of K525222 and BBa_E1010 without TAT-sequence and lipid anchor mostly found in the cytoplasm. The periplasmatic isolation does destroy few complete cells, therefore in the periplasmatic fraction sequence coverage is found. The gel lanes in the periplasmatic fraction are clearly less intense, the protein concentration is very low compared to the lysis fraction. Sequence coverage in the media fraction shows, that the protein is probably secreted into the media.